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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 83-92
Sustained Gene Expression in Retrovirally Transduced, Engrafting
Human Hematopoietic Stem Cells and Their Lympho-Myeloid Progeny
By
Linzhao Cheng,
Changchun Du,
Catherine Lavau,
Shirley Chen,
Jie Tong,
Benjamin P. Chen,
Roland Scollay,
Robert G. Hawley, and
Beth Hill
From SyStemix, Inc, Palo Alto, CA; the Oncology Gene Therapy Program,
The Toronto Hospital, Toronto; and the Department of Medical
Biophysics, University of Toronto, Toronto, Ontario, Canada.
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ABSTRACT |
Inefficient retroviral-mediated gene transfer to human hematopoietic
stem cells (HSC) and insufficient gene expression in progeny cells
derived from transduced HSC are two major problems associated with
HSC-based gene therapy. In this study we evaluated the ability of a
murine stem cell virus (MSCV)-based retroviral vector carrying the
low-affinity human nerve growth factor receptor (NGFR) gene as reporter
to maintain gene expression in transduced human hematopoietic cells.
CD34+ cells lacking lineage differentiation markers
(CD34+Lin ) isolated from human bone marrow
and mobilized peripheral blood were transduced using an optimized
clinically applicable protocol. Under the conditions used, greater than
75% of the CD34+ cell population retained the
Lin phenotype after 4 days in culture and at least 30%
of these expressed a high level of NGFR (NGFR+) as
assessed by fluorescence-activated cell sorter analysis. When these
CD34+LinNGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term
stromal cultures, sustained reporter expression was observed in
differentiated erythroid and myeloid cells derived from transduced
progenitors, and in differentiated B-lineage cells after 6 weeks.
Moreover, when these transduced CD34+LinNGFR+ cells were
used to repopulate human bone grafts implanted in severe combined
immunodeficient mice, MSCV-directed NGFR expression could be detected
on 37% ± 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the
MSCV retroviral vector in the development of effective therapies
involving gene-modified HSC.
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INTRODUCTION |
HEMATOPOIETIC STEM CELLS (HSC) provide an
attractive target for somatic cell-based gene therapy because they have
the potential to continue producing progeny cells containing a
therapeutic gene indefinitely. Hematological diseases that could
benefit from HSC-based gene therapy approaches include hereditary
diseases as well as other diseases such as acquired immunodeficiency
syndrome and cancer.1 Retroviral vectors, which are being
used in the majority of current clinical trials, are a primary choice
as the vehicle for gene delivery. They are capable of integrating into
cellular chromosomes, resulting in stable transmission to every progeny cell derived from transduced HSC.2-4 However, it has become
clear that current protocols for transducing human HSC with retroviral vectors are inefficient.5,6 Although transgenes in
engrafting cells have been detected using sensitive assays such as
polymerase chain reaction (PCR), they have rarely been found in
long-term repopulating cells.5-7 These results are in
striking contrast to the efficient transduction of less primitive human
progenitors, which are able to form colonies under in vitro culture
conditions,7 and of mouse HSC.8
Therefore, it is important to directly assess gene transduction into
rare HSC capable of repopulating in vivo and of generating multiple
(myeloid and lymphoid) lineages of differentiated hematopoietic cells.
Human HSC are included in a rare population of cells that bear the CD34
surface antigen (CD34+) and lack all lineage
differentiation markers (Lin). However, only a small
fraction of CD34+Lin cells have HSC
activities, operationally defined by long-term in vivo marrow
repopulating activity and the ability to give rise to both myeloid and
lymphoid progeny.9,10 Several animal models have been
developed in an attempt to detect HSC among the
CD34+Lin population. One of these is the
severe combined immunodeficient-human (SCID-hu) bone
system, in which a human fetal bone fragment is implanted in SCID mice
as a supportive human hematopoietic microenvironment.11,12 Using this assay, it was found that the in vivo marrow repopulating activities of CD34+Lin cells mainly
resided in a subset of cells expressing the Thy-1 antigen
(Thy-1+CD34+Lin).12,13
In contrast,
Thy-1CD34+Lin cells
were found to lack in vivo SCID-hu repopulating activity, although they
were enriched for cells with in vitro colony-forming potential.12,13 Analogous results have been obtained using other SCID mouse models and a human-fetal sheep model.14,15 Thus, SCID repopulating cells (SRC) are considered to be more primitive
than hematopoietic progenitor cells with in vitro activities, making
the surrogate SCID-hu system a useful small animal assay to distinguish
candidate human HSC from hematopoietic progenitors. In a recent report
it was found that SRC within the CD34+ population from
human bone marrow (BM) were rarely transduced (<1%) even though
retroviral-mediated gene transfer to colony-forming progenitors was
highly efficient (up to 95% gene marking) under the conditions
used.14 The inability to efficiently transduce SRC in that
study mirrors the low level of HSC transduction observed with existing
protocols in various human gene therapy trials.1,5-7
The second potential problem associated with retroviral vector-based
gene therapy is transcriptional silencing of the introduced transgene.
Retroviral vectors derived from Moloney murine leukemia virus (MoMLV)
are the most commonly used retroviral vectors in clinical
trials.2-4 In a standard configuration, the gene of interest is placed under the transcriptional control of the viral long
terminal repeat (LTR) because gene expression driven by the LTR is
generally higher than when the exogenous gene is under the control of
an internal promoter.16,17 However, it has been reported
that MoMLV LTR-mediated gene expression is frequently downregulated
during differentiation of HSC and inactive in several cell
types.17-19 Because the LTR of the murine stem cell
virus (MSCV) retroviral vector is permissive for expression in murine
HSC,8,20 we were interested in examining the performance of
the MSCV vector in candidate human HSC as well as in their
differentiated progeny.
Recently, a number of groups have used various cell-surface molecules,
including murine CD24 (HSA), murine CD8a (Lyt-2), and the low-affinity
human nerve growth factor receptor (NGFR), to measure efficiency of
gene transfer into hematopoietic precursors and to follow transgene
expression in their marked progeny.17,21,22 Retroviral
vectors encoding NGFR have been used successfully by others to
transduce human T lymphocytes and hematopoietic cells, and no adverse
effects on the transduced cells have been observed.22 Therefore, we decided to use the human NGFR gene as a selectable marker
and reporter in this study. Multiparameter flow cytometric analysis
allowed cell-surface expression of NGFR to be easily monitored in
various hematopoietic cell populations defined by cell-surface markers
(either CD34+Lin cell populations or
differentiated cells belonging to a particular lineage). Similar
efficiencies of gene transfer into colony-forming progenitors were
obtained by a MSCV-based vector as for a MoMLV-based vector. However,
we found that NGFR transgene expression mediated by MSCV LTR was
substantially higher in differentiated erythroid, myeloid, and
B-lymphoid progeny than that mediated by MoMLV LTR. Based on these
findings we evaluated persistence of MSCV LTR-directed expression in
progeny derived from transduced SRC. We show that our protocol for
transducing SRC with the MSCV-based vector was efficient, and that a
high percentage of transduced human cells continued to express NGFR
after long-term reconstitution of SCID-hu bone mice.
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MATERIALS AND METHODS |
Construction and detection of retroviral expression vectors.
The LXSN type of MoMLV vector was used as the parental vector for
LINGFR.23 After deleting the internal SV40 promoter and the
neo gene in LXSN, an internal ribosome entry site (IRES) from the encephalomyocarditis virus was inserted.24 The human
(p75) NGFR gene was then placed after the IRES.25 The
resultant vector was named LINGFR to reflect the order of essential
components (LTR-IRES-NGFR). The MINGFR vector was similarly constructed
by replacing the neo gene driven by an internal promoter in
MSCVneoEB with the IRES-NGFR cassette from LINGFR.20 Gene
expression mediated by the MINGFR vector is directed by the MSCV LTR,
whereas in LINGFR gene expression is driven by the MoMLV LTR. A third
vector, MINT, was created by truncating the NGFR gene in MINGFR after
the transmembrane domain (at the Nae I site). All the plasmids
were purified and used in packaging cell transfections as
described.26
The primers used to amplify transgene-specific (IRES) DNA sequences
common to all three vectors were: upstream, 5 -CGT TAC TGG CCG
AAG CCG CT-3; and downstream, 5-AAC CTC GAC TAA ACA CAT
GT-3. The primers used to amplify the endogenous human
 -globin sequence of genomic DNA as a control for PCR assays were:
upstream, 5-ACA CAA CTG TGT TCA CTA GC-3; and downstream,
5-CAA CTT CAT CCA CGT TCA CC-3. Forty-cycle PCR reactions
for both target sequences were performed with an annealing temperature
of 62°C in the presence of 1.5 mmol/L MgCl2. PCR
products (a 485-bp IRES-specific fragment and a 220-bp
-globin-specific fragment) were separated by 4% agarose gel
electrophoresis.
Specific antibodies for fluorescence-activated cell sorting (FACS)
analyses.
A hybridoma producing mouse IgG1 monoclonal antibody (MoAb) against the
human NGFR was obtained from the American Type Culture Collection (ATCC
HB8737; Rockville, MD). Purified antibodies from mouse
ascites were conjugated either directly with fluorescein isothiocyanate
(FITC) or with R-phycoerythrin (PE) after deleting the Fc fragment. A
CD34 antibody (Tuk3) conjugated with sulfo-rhodamine (SR), and a panel
of FITC-conjugated mouse MoAbs against lineage differentiation markers
were used to isolate and analyze transduced cells.27 This
lineage panel (collectively called Lin) comprised CD2, CD4, CD14, CD15,
CD16, CD19 (Becton Dickinson, San Jose, CA) and glycophorin A
(Immunotech, Westbrook, ME). An FITC-conjugated antibody (MA2.1, ATCC
HB54) against HLA-A2 was used to identify and monitor
MA2.1+ donor cells in SCID-hu bone mice.12
Propidium iodide (0.5 µg/mL) was added to cell suspensions after
antibody staining to exclude dead/dying cells from FACS analyses. FACStarPlus or FACS Vantage cell sorters (Becton Dickinson)
equipped with a primary Agron laser and a dye-laser (required for
detecting SR signals) were used for cell sorting. FACScan analyzers
(Becton Dickinson) equipped only with an Argon laser were used to
phenotype harvested cells after in vitro and in vivo assays.
Cytokines, media, and cell lines.
Recombinant human interleukin-3 (IL-3), IL-6, granulocyte-macrophage
colony-stimulating factor (GM-CSF), steel factor (SLF, also called stem
cell factor), and leukemia inhibitory factor (LIF) were obtained from
Sandoz Pharma (Basel, Switzerland), and erythropoietin (Epo) was
purchased from Amgen (Thousand Oaks, CA). Dulbecco's modified Eagle's
medium (DMEM), Iscove's modified Dulbecco's medium
(IMDM), and RPMI 1640 culture media were purchased from
GIBCO-BRL (Gaithersburg, MD) and fetal calf serum (FCS) from Hyclone
(Logan, UT). TF1 cells (ATCC CRL-2003) were maintained in RPMI 1640 plus 10% FCS and 2 ng/mL GM-CSF.28
Production of retroviral supernatants and transduction protocol.
Amphotropic supernatants produced by the human 293 (embryonic kidney
fibroblast) cell-based ProPak-A packaging line were made through
transduction with VSV-G pseudotyped viral stocks as
described.29,30 NGFR-expressing ProPak-A cells were
enriched by flow cytometry sorting and expanded in culture. Amphotropic
supernatants were then collected from stable ProPak-A producers,
filtered, and stored at  80°C until use. For transduction,
fresh or previously frozen vector supernatants were mixed at a 1:1
ratio with media containing target cells in the presence of 8 µg/mL
polybrene (Sigma, St Louis, MO). The transduction mixture was then
centrifuged at 1,800g at 32°C to 35°C for inoculation.
After the 4-hour "spinoculation," the cells were washed once and
cultured in appropriate media.
Isolation and transduction of human hematopoietic progenitors.
Human BM aspirates and mobilized peripheral blood (mPB, collected at
day 4 or 5 after G-CSF treatment) were obtained from healthy donors in
compliance with regulations established by the federal and state
governments. Low-density (<1.077 g/cm3) mononuclear BM
cells after Ficoll-Hypaque gradient (Pharmacia, Piscataway, NJ) or
apheresed mPB cells were stained with a CD34 antibody included in the
Isolex kit (Baxter Biotech Immunotherapy Division, Irving, CA).
CD34+ cells were magnetically isolated by Isolex using a
modified protocol developed at Systemix.27 The purity of
CD34+ cells from both BM and mPB was usually greater than
90% (n = 10 for BM and n = 20 for mPB). These isolated cells were then stained with CD34-SR and Lin-FITC, and
CD34+Lin cells were sorted by FACS and
activated ex vivo for gene transduction. Cells ( 106/mL)
were cultured overnight in IMDM/RPMI 1640 (1:1) medium plus 10% FCS
supplemented with 10 ng/mL IL-3 and IL-6, and 100 ng/mL SLF. The next
day cells were transduced with ProPak-A viral supernatants for 4 hours
as described above. The transduction procedure was repeated the
following day, and then the medium was changed and the cells were
cultured for additional 2 days to allow gene expression.
In vitro progenitor assays.
Methylcellulose and reagents for clonogenic progenitor assays were
obtained from StemCell Technologies (Vancouver, Canada). Cells (4 × 102) were added to 1 mL methylcellulose medium
(MethoCult H4230) supplemented with IL-3, IL-6, GM-CSF (10 ng/mL each),
SLF (100 ng/mL), and Epo (2 U/mL). Cell mixtures were plated in 35-mm
suspension culture dishes (Nunc, Roskilde, Denmark), and
incubated at 37°C. After 2 weeks, colonies (>100 cells) were
enumerated in each of three triplicate plates. Subsequently some
individual colonies were randomly picked. Cells were then lysed and an
aliquot was used for a 40-cycle PCR analysis to detect specific DNA
sequences. Colonies which yielded a positive signal for the human
-globin sequence were included in the calculations of colony-forming
cell (CFC) gene transfer efficiency. Colonies which also yielded a transgene signal equal to or stronger than the -globin signal in the
same PCR reaction were considered to be positive for the transgene. The
remaining cells were obtained in bulk at the end of 2-week CFC assays
for FACS analysis of NGFR expression. Cells were washed three times
with phosphate-buffered saline plus 0.5% human IgG (Gammimmune, Miles
Inc, Elkhart, IN) before being stained with a PE-conjugated NGFR
antibody and FITC-conjugated antibodies against glycophorin A, CD14, or
CD15.
For stromal-dependent cobblestone area-forming cell (CAFC)
assays,31 a stromal cell line (SyS-1) derived from murine
BM was used.13 Cultures were maintained with IMDM/RPMI 1640 (1:1) medium plus 10% FCS supplemented with 10 ng/mL IL-6 and 50 ng/mL LIF. Up to 100 cells were cultured on sub-confluent monolayers of SyS-1
stromal cells in each well of 96-well plates. Half of the medium was
replaced weekly, and the cultures were monitored for 6 weeks. CAFC
frequencies were calculated based on the results obtained at week 5. Total cells were procured and pooled from wells which contained at
least one cobblestone area at week 6. After being filtered through a
30-µm mesh filter, cells were stained as described before, with the
PE-conjugated NGFR antibody and the FITC-conjugated CD19 antibody.
In vivo SCID-hu bone assays.
Immunodeficient C.B-17 scid/scid (SCID) mice were used as
recipients of human fetal bone fragments to construct the SCID-hu bone
mice.11,12 In each of two independent experiments, bone fragments were derived from the same human fetal tissue, which was
negative for HLA-A2 and B17 and was not recognized by the corresponding
MoAb MA2.1 (ATCC HB-54). Eight weeks after bone implantation, SCID-hu
bone mice were used as recipients of transduced human hematopoietic
precursors whose HLA-type was MA2.1+. The transduced donor
cells were injected directly into the implanted bones
(MA2.1) residing in SCID mice according to the
published protocol.12 Fifty thousand to 100,000 cells
(based on CD34+Lin cell counts) from the
same cell population were injected into each of two bone fragments
implanted in SCID mice. Nine weeks after injection, recipient mice were
terminated, the implanted bone fragments were surgically removed, and
total cells from BM were obtained. Then harvested cells were then
stained with the PE-conjugated NGFR antibody and the FITC-conjugated
MA2.1 antibody. Propidium iodide was added after antibody staining to
cell suspensions to exclude dead/dying cells from FACS analyses. Using
a FACScan analyzer, levels of engraftment (based on the presence of
MA2.1+ donor cells) and transgene expression (based on the
presence of NGFR on the surface of donor cells) were
assessed by FACS.
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RESULTS |
Transduction of human TF1 hematopoietic progenitor cell line with MSCV-
and MoMLV-based vectors encoding NGFR.
Several retroviral vectors containing the NGFR gene as a reporter were
constructed to compare gene transfer and expression in human
hematopoietic cells (Fig 1). NGFR
expression is directed by the MSCV LTR in MINGFR and MINT, or directed
by the MoMLV LTR in the LXSN-derived vector LINGFR.20,23 To
achieve a higher efficiency of transduction of human hematopoietic
precursors than has been routinely accomplished with recombinant
retroviral stocks prepared using conventional murine packaging lines,
amphotropic retroviral vector supernatants were produced using a new
human 293 cell-based packaging line (ProPak-A).29
Transduction efficiencies were first assessed on the growth
factor-dependent human CD34+ progenitor line
TF1.28 As shown in Fig 2A,
60% of TF1 cells were transduced by either MINGFR or LINGFR vector
4 days after the cells were exposed to 50% (vol/vol) of viral
supernatants in a "spinoculation" protocol. Subsequent limiting
dilution experiments with MINGFR and LINGFR vector preparations
established that the two viral stocks had comparable titers (data not
shown). Moreover, as indicated by the mean fluorescence intensities of
the positive peaks, both vectors directed similar NGFR expression
levels in TF1 cells (Fig 2A). The two vectors also performed equally
well when tested on other cell lines such as murine NIH3T3 fibroblasts (data not shown).
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| Fig 1.
Schematic representation of NGFR-encoding retroviral
vectors. The LXSN type of MoMLV vector was used as the parental vector backbone in the construction of LINGFR and the MSCVneoEB vector was
used to derive the MINGFR and MINT vectors. In all cases, a NGFR gene
has been placed downstream of an IRES, replacing the previous
neo genes driven by internal promoters. Inclusion of the IRES
potentially allows for coexpression of an upstream gene on bicistronic
transcripts which also encode the NGFR reporter. The LTR of MoMLV
directs NGFR gene transcription in LINGFR whereas the MSCV LTR is used
to express the NGFR gene in MINGFR and MINT. The NGFR gene in MINT was
truncated after the transmembrane (TM) domain.
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| Fig 2.
FACS analysis of NGFR expression in retrovirally
transduced TF1 cells. (A) Histograms of TF1 cells expressing the NGFR
transgene after transduction with the LINGFR, MINGFR, or MINT vectors.
TF1 cells were transduced with equal volumes of amphotropic viral supernatants for 4 hours (as described in Materials and Methods) and
NGFR expression was analyzed by FACS 4 days later after staining for
cell-surface NGFR with a FITC-conjugated anti-NGFR antibody. The
profile of nontransduced TF1 cells (thin lines representing 0.5%) was
overlaid to highlight transduced cell NGFR+ populations.
Approximately 62%, 59%, and 40% of TF1 cells were transduced with
the LINGFR, MINGFR, and MINT vectors, respectively. (B) Kinetics of
NGFR expression in TF1 cells after transduction with the MINT vector.
After 4-hour exposure to MINT vector supernatant, TF1 cells were either
processed immediately (day 0) or cultured for 1, 2, 3, or 4 days and
then stained for NGFR expression. Based on FACS histograms as shown in
(A), the percentages of NGFR-expressing cells and relative levels of
NGFR expression in transduced cells were plotted as a function of time
in culture. The relative intensity of NGFR expression is defined by
increased mean fluorescence intensity (MFI) normalized by the MFI of
nontransduced cells.
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A third vector, MINT, in which the NGFR gene in the MINGFR vector was
C-terminally truncated after the transmembrane domain, was used to
examine the kinetics of NGFR expression after viral transduction. The
titer of MINT viral stock was somewhat lower and 40% TF1 cells were
transduced and expressed NGFR on cell surface after 4 days (Fig 2A). To
determine the kinetics of NGFR transgene expression after transduction,
MINT-transduced TF1 cells were either stained for NGFR surface protein
immediately posttransduction or cultured for various periods of time
and then analyzed as described above. No NGFR signal could be detected
on the cell surface immediately after the transduction protocol (Fig
2B). Subsequently, the percentage of NGFR-expressing
(NGFR+) cells as well as NGFR signal intensities
progressively increased with time, reaching a maximum of 40% 4 days
posttransduction (Fig 2A and B). Afterward the values of these two
parameters remained constant for at least 2 months if transduced TF1
cells were maintained in a proliferating phase. The doubling time of
TF1 cells is 20 hours; thus, it took a period equivalent to three
cell divisions for continuously proliferating TF1 cells to reach the
maximal and stable level of the NGFR surface signal. This finding is
consistent with accumulated data that efficient retroviral-mediated
expression (transcription and translation) occurs only after
integration into cellular chromosomes which, in the case of C-type
retroviruses, is dependent on cell division.
The MINT vector was initially constructed to minimize the possibility
of NGFR functioning as a signal transducer in hematopoietic cells.
However, no adverse effect was observed due to full-length NGFR
expression on the growth of transduced TF1 cells (data not shown) or
primary human hematopoietic cells assayed in vitro (see below). Because
the titer of the MINT vector was somewhat lower than that of MINGFR or
LINGFR vector (Fig 2A), we restricted our attention solely to the
latter two vectors to compare directly the expression properties of
MSCV- and MoMLV-based retroviral vector backbones after transduction of
primary human hematopoietic precursors.
Transduction of human CD34+Lin cells.
Enriched CD34+ cells from BM or mPB were further purified
by FACS to reach greater than 95% purity with respect to CD34
expression and lack of expression of lineage-specific differentiation
markers (CD2 and CD4 for T cells, CD16 for natural killer [NK] cells, CD19 for B cells, CD14 for monocytes, CD15 for granulocytes, and glycophorin A for erythroid cells). These highly purified cell populations (denoted as CD34+Lin) were
prestimulated in a cytokine cocktail (IL-3, IL-6 plus SLF) overnight
and then exposed to retroviral supernatants in the
"spinoculation" protocol. Viral supernatants were added on days 1 and 2, and then the cells were cultured for 2 more days to allow gene
expression. Gene transfer efficiency and NGFR expression level were
then investigated by FACS analysis. Under the conditions used, total
cell numbers increased during this period by approximately fourfold or
twofold for input CD34+Lin cells
isolated from BM (n = 3) or mPB (n = 6), respectively. Shown in
Fig 3 as an example, 80% of the cells
retained the CD34+Lin phenotype after 4 days in culture while the remaining cells lost the CD34 antigen and
gained one or more lineage differentiation markers (n = 3 for mPB). The
CD34+ content of the cultures decreased rapidly thereafter,
concomitant with the continued increase in total cell numbers. Among
mPB cells which retained the CD34+Lin
phenotype (gated in the R2 region in Fig 3), greater than 30% expressed the NGFR transgene at day 4, irrespective of which retroviral vector backbone was used. The percentages of NGFR+ cells
and the corresponding mean fluorescence intensities were, however,
marginally but consistently higher for cells transduced by MINGFR than
for those transduced by LINGFR (n = 3 for mPB). Similar results were
obtained with transduced CD34+Lin-cells of BM origin (n = 2, data not shown).
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| Fig 3.
FACS analyses of NGFR transgene expression in transduced
CD34+Lin cells. Sorted human mPB
CD34+Lin cells were activated and
transduced with LINGFR or MINGFR vector supernatants. Two days after
viral transduction (4 days in culture), expression of CD34 (stained
with an SR-conjugated antibody) and the Lin markers (stained with
FITC-conjugated antibodies) were analyzed (dot plots in upper panels).
Live cells which retained the CD34+Lin
phenotype were gated (R2 regions) and percentages of gated cells among
the total live cells were determined (80% in all cases). Efficiencies of gene transfer and expression levels in these
CD34+Lin cell populations were assessed by
presence of NGFR (stained by a PE-conjugated antibody) and are plotted
as histograms in the lower panels. The gates set up to sort cells
expressing NGFR (NGFR+, R4) and cells lacking the NGFR
surface reporter (NGFR, R3) are indicated as are the
percentages of NGFR+ cells.
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Transduction of human CFC and CAFC progenitors.
At day 2 posttransduction (4 days in culture), cells were plated in
methylcellulose to detect clonogenic myeloid and erythroid progenitors
in a standard CFC assay. Total numbers of colonies were first
enumerated by visual inspection and then the efficiency of gene
transduction was assessed either by PCR to detect the presence of the
vector sequences in individually picked colonies or by FACS to examine
NGFR expression in populations of CFC-derived myeloid and erythroid
cells. Approximately 25% of the input cells transduced by either
MINGFR or LINGFR formed colonies, similar to nontransduced controls
(Table 1). PCR analysis indicated that 28%
(out of 64 picked CFC colonies) were transduced by MINGFR and 34% (out
of 65 picked CFC colonies) were transduced by LINGFR, indicating that
both vectors were capable of achieving similar efficiencies of gene
transfer to CFC. When NGFR expression in differentiated cells derived
from CFC was analyzed after 2 weeks in culture, we found that only
7.7% to 12.5% of the total cell populations expressed detectable cell
surface NGFR (Table 1).
We next sorted transduced CD34+Lin cells
into two fractions on the basis of NGFR expression at day 2 posttransduction (Fig 3). The fact that a small percentage of
NGFR cells were capable of generating
NGFR+ differentiated cells in the CFC assays (by FACS, data
not shown) illustrated that not all of the retrovially transduced cells
expressed sufficient levels of NGFR transgene at day 2 posttransduction to permit their identification and isolation. Thus, the frequencies of
NGFR+ cells at this time point (day 2 posttransduction)
were presumed to be underestimates of the actual values of cells
containing the transgene. Nonetheless, since enrichment for
cell-surface NGFR greatly simplified functional analyses of transduced
cells and studies of LTR-mediated gene expression in functionally
heterogeneous CD34+Lin cells, we
subsequently focused on those NGFR+ subpopulations that
were clearly transduced and could be separated from untransduced cells.
Approximately 20% of the
CD34+LinNGFR+ cells
generated by transduction with MINGFR or LINGFR formed CFC colonies
(Table 1), a rate slightly less than that of unsorted cells (which is
25%). Based on these plating efficiencies (20% v 25%),
this result would indicate that the majority of transduced CFC
progenitors by both vectors also expressed the NGFR transgene 2 days
posttransduction. Interestingly, however, NGFR transgene expression in
CFC-derived cells at the end of the 2 week CFC assay period are
different among NGFR+ cells transduced by the two vectors.
Whereas 78% of CFC-derived progeny cells derived from
MINGFR-transduced, sorted
CD34+LinNGFR+ cells
continued to express the NGFR transgene after 2 weeks, approximately
one half of cells derived from LINGFR-transduced NGFR+ CFC
had completely lost NGFR transgene expression while the remaining expressed NGFR at a reduced level (Table 1 and
Fig 4). PCR analysis after cell sorting of
LINGFR-transduced, CFC-derived cells which did not express NGFR after
2-week CFC assays confirmed that the transgene was still present in
this NGFR differentiated cell population (data not
shown). Further multiparameter FACS analyses shown in Fig 4 indicated
that downregulation of LINGFR-mediated NGFR expression occurred in
differentiated erythroid (glycophorin A+) cells and
granulocytes (CD15+), as well as in (CD14+
cells) monocytes (data not shown). The downregulation of MoMLV LTR-mediated NGFR expression by the LINGFR vector in CD14+
and CD15+ myeloid cells was also seen in a suspension
culture assay (data not shown). These findings are consistent with
those of a recent report in which the MoMLV LTR-mediated transcription
is downregulated in differentiated erythroid/myeloid progeny derived
from transduced CD34+ cells in CFC and suspension cultures,
and the MSCV LTR-mediated transcription is substantially
higher.19
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| Fig 4.
LTR-mediated NGFR transgene expression in progeny of
transduced CFC. Sorted NGFR-expressing,
CD34+Lin cells transduced with either the
LINGFR (LINGFR+) or MINGFR (MINGFR+)
vectors (see Fig 3) were assayed for CFC activity. After 14 days CFC
numbers were enumerated (shown in Table 1), and total cells were
obtained and stained for lineage markers and NGFR transgene expression.
Fifty-two percent and 88% of erythroid cells (glycophorin A+), and 71% and 91% of granulocytes
(CD15+) derived from LINGFR+ and
MINGFR+ CFC, respectively, retained the NGFR reporter on
cell surface.
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Because silencing of MoMLV LTR-mediated expression had previously been
observed in resting human T cells,17 and in differentiated erythroid/myeloid cells as shown above, we next examined whether this
phenomenon also occurred in differentiated CD19+ B-lineage
cells. The sorted NGFR+ and NGFR
fractions of CD34+Lin cells (lacking
CD19 expression) were plated at limiting dilution on stromal cell
monolayers in a CAFC assay. There are two unique aspects of this
long-term stromal-dependent CAFC assay compared with CFC assays: (1)
CD19+ B-lineage cells as well as myeloid cells are
generated from CD34+Lin cells; and (2)
late-appearing (after 5 weeks) cobblestone areas are considered to be
derived from progenitors which are more primitive than
CFC.13,31 The frequencies of CAFC in the various transduced cell populations were calculated based on Poisson distribution (Fig 5A). The frequencies of CAFC present
in the NGFR+ subpopulations transduced by the MINGFR and
LINGFR vectors were similar. However, both frequencies were more than
10-fold lower than the values calculated for the corresponding
NGFR subpopulations, indicating that the majority of
CAFC were either not transduced at all or not expressing NGFR transgene
2 days posttransduction. An additional experiment using different
preparations of viral supernatants confirmed this finding (data not
shown).
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| Fig 5.
Maintenance of NGFR transgene expression in CAFC progeny.
(A) Sorted NGFR-expressing, CD34+Lin cells
transduced with either the LINGFR (LIN, NGFR+) or MINGFR
(MIN, GFR+) vectors, or the sorted
CD34+Lin cells lacking NGFR expression at
day 2 posttransduction with LINGFR (LIN, NGFR) or MINGFR
(MIN, NGFR) vectors (see Fig 3) were plated on SyS-1
stromal cell monolayers. Two-fold serial dilution of 100 CD34+Lin input cells were seeded per well
and examined for cobblestone area (CA) formation weekly up to 6 weeks.
The number of wells lacking any CA at week 5 were plotted as a function
of numbers of sorted cells, respectively. The frequencies of CAFC were
estimated based on Poisson distribution and the results are indicated.
(B) NGFR transgene expression in B cells formed at week 6 of CAFC assays. CD19+ B cells (in addition to CD14+
and CD15+ myeloid cells) were generated from sorted
CD34+LinNGFR+ cells in the
presence of SyS-1 stromal cells as shown by the presence of the CD19
marker. Among CD19+ B cells, approximately 14% and 50%
of the progeny cells derived from LINGFR-transduced
(LINGFR+) and MINGFR-transduced (MINGFR+)
CD34+LinNGFR+ cells,
respectively, expressed the NGFR transgene at the end of CAFC assays.
|
|
NGFR transgene expression was examined in differentiated B cells at
week 6 of CAFC assays. Cells in cobblestone area-containing wells
initially seeded with preselected NGFR+ transduced cells
were obtained and pooled for simultaneous FACS analysis of the CD19
B-cell marker and NGFR transgene expression (Fig 5B). The majority of
harvested hematopoietic cells were myeloid cells. A population of
CD19+ cells was detected in pooled CAFC+ wells
containing transduced cells by either LINGFR or MINGFR vector, or
mock-transduced cells (Fig 5B). Although 50% of CD19+
cells derived from MINGFR-transduced
CD34+LinNGFR+ cells
expressed a high level of NGFR reporter, a low to medium level of
cell-surface NGFR could only be detected on 14% of
CD19+ cells derived from LINGFR-transduced
CD34+LinNGFR+ cells. Taken
together, the results show that the MSCV LTR appears to be less
susceptible to transcriptional silencing mechanisms than the MoMLV LTR
in multiple lineages during in vitro differentiation of transduced
human hematopoietic precursors.
Transduction of candidate human HSC assayed in SCID mice.
It has been shown that subsets of
CD34+Lin cells capable of long-term
(>8 weeks) engraftment of fetal human bone implants in SCID mice
(SCID-hu bone assay) exhibit B-lymphoid and myeloid potential as well
as secondary repopulating capacity.12,13 To assess whether
transduced CD34+Lin cells expressing the
NGFR reporter have candidate HSC activity, transduced mPB cells were
tested in the SCID-hu bone assay for the presence of SCID repopulating
cells (SRC). Because NGFR expression is higher in progeny derived from
CD34+Lin cells transduced with MINGFR
than with LINGFR in the in vitro assays described above, for SRC assays
we restricted our attention to the MINGFR-transduced cells. Nine weeks
postinjection, the presence of donor (MA2.1+) cells and the
NGFR transgene expression was examined by FACS analysis of total cells
obtained from the human bone implants (Fig
6). Cell-surface expression of NGFR could not be detected in
nontransduced human hematopoietic cells (Fig 6A and B). The sorted
CD34+LinNGFR+ cells that had
been transduced by the MINGFR vector readily engrafted and maintained
NGFR transgene expression in donor cells 9 weeks postinjection (Fig 6C
and D).
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| Fig 6.
Representative FACS analyses of NGFR transgene expression
in progeny of MINGFR-transduced, CD34+Lin
cells recovered from SCID-hu bone mice. Harvested cells from implanted
human bone fragments were stained with the FITC-conjugated MA2.1
antibody recognizing the donor cell's HLA, and the PE-conjugated anti-NGFR antibody recognizing the transgene expression on cell surface. Viable cells were then collected and analyzed by FACS. Bone
fragments in the absence of injected cells (A) and the presence of
mock-transduced cells (B) were included as controls to distinguish donor cells (MA2.1+) from endogenous human cells
and/or contaminating murine cells (MA2.1). Cells
recovered from two different bone implants injected with sorted
CD34+LinNGFR+ cells
transduced with MINGFR are shown in (C) and (D). Approximately 35.5%
(C) and 44.7% (D) of donor-derived cells were expressing the NGFR
reporter after 9 weeks in vivo. See Materials and Methods for
experimental details and Table 2 for a summary.
|
|
The results of our first two experiments are summarized in
Table 2. Nine of 10 injections with
mock-transduced cells showed donor cell engraftment. Because this
frequency (90%) is similar to historic data obtained using freshly
isolated human hematopoietic precursors,12 we believe that
our protocol for ex vivo cell culture and transduction did not
significantly alter the long-term SRC potential of
CD34+Lin cells. Both NGFR+
and NGFR subpopulations selected at day 2 posttransduction by MINGFR engrafted (5 of 5 and 2 of 2, respectively).
All 5 bone grafts that successfully repopulated with MINGFR-transduced,
preselected CD34+LinNGFR+
cells showed reasonable levels of NGFR transgene expression (28% to
45% [37% ± 6%, n = 5] of the donor cells). In one experiment where sufficient numbers of donor-derived cells were available for an
additional FACS analysis, both CD19+ (B-lineage) and
CD19NGFR+ (transduced donor) cells were
found (data not shown). These findings showed that the MSCV LTR is
functional in primitive human hematopoietic precursors with SRC
potential and remains active for long periods in their transduced
progeny after in vivo differentiation.
| |
DISCUSSION |
In this report we evaluated the MSCV-based MINGFR vector carrying the
NGFR reporter gene for its ability to efficiently transduce hematopoietic precursors purified from adult human BM or mPB. Based on
FACS analyses and in vitro functional assays, similar efficiencies of
gene transfer into CD34+Lin cells as
well as CAFC and CFC progenitors were observed as for the MoMLV-based
LINGFR vector. Moreover, stable gene transfer into HSC (or SRC), based
on the NGFR reporter expression in engrafted donor cells, was observed
in 7 of 7 grafts of MINGFR-transduced cells. Because of the qualitative
nature of the SCID-hu mouse model (in the absence of a limiting
dilution analysis which requires large numbers of engineered animals),
it is difficult to estimate the frequencies of HSC/SRC-based gene
transfer by MINGFR, and to what extent the improved retroviral vector
stocks and the "spinoculation" transduction protocol used in this
study contributed to the success of these experiments. Because others
reported recently that inclusion of certain cytokines (eg, Flk2/Flt3
ligand) and the presence of cell extra-cellular matrix molecules (eg,
fibronectin fragments) can further preserve/activate candidate HSC and
increase efficiencies of gene transfer into them, we expect that the
transduction protocol reported here can be further optimized with these
molecules.14,32,33
Although the level of NGFR expression directed by the LTR of either
vector was comparable in several cell lines and in the total
CD34+Lin cell populations shortly after
transduction, NGFR transgene expression mediated by MSCV LTR is
substantially higher than that directed by MoMLV LTR in differentiated
progeny derived from transduced CD34+Lin
cells (Figs 4 and 5B). We observed that the LTR-mediated gene expression from LINGFR (which is based on the LXSN-type of MoMLV vector) was downregulated in differentiated cells belonging to multiple-lineages including erythroid, myeloid, and B-lymphoid cells.
Similar observations with MoMLV vectors have been made by other
investigators in erythroid/myeloid lineages,19 in T cells,17 and in mouse hematopoietic cells after in vivo BM
repopulation.18 Moreover, the phenomenon of the in vivo
downregulation of gene expression of MoMLV LTR (typically caused by
transcriptional silencing/inactivation) is not limited to the
hematopoietic system, as it has also been observed in transduced
primary fibroblasts, myoblasts, and hepatocytes in a variety of animal
models.34 However, it should be noted that others have
documented MoMLV LTR-directed gene expression in the T-cell or myeloid
progeny of transduced CD34+ cells purified from cord blood
or fetal liver after repopulation of SCID-hu thymus or SCID-hu bone
grafts.35-37 Nonetheless, the possible extinction of MoMLV
LTR-mediated expression needs to be taken into account when sustained
expression in multiple myeloid and lymphoid lineages is deemed
necessary for therapeutic benefits in HSC-based gene transfer
applications.
Based on the outcome of this study, it would appear that MSCV-based
vectors may offer advantages over conventional MoMLV-based vectors for
gene delivery to the human hematopoietic system. The ability to direct
sustained high-level expression of exogenous genes in differentiated
cells derived from transduced HSC/progenitors is obviously a desirable
goal of a number of human gene therapy protocols targeting congenital
blood disorders.1 In addition, a vector that is permissive
for expression in HSC may be of utility in those cancer gene therapy
applications where the intent is to confer a drug-resistant phenotype
to the patient's mature hematopoietic cells and their precusors to
augment the therapeutic index of high-dose anti-tumor chemotherapy. In
this regard, other types of retroviral vectors are currently being
investigated for this purpose and are promising in human progenitor
cells assayed in vitro.38 It remains to be determined
whether these vectors are also functional in directing gene expression
in HSC/SRC and whether they are more active than MSCV.
The sensitivity of the NGFR reporter system allowed facile monitoring
of transgene expression during differentiation of transduced human
hematopoietic precursors into progeny cells belonging to multiple
(myeloid and lymphoid) lineages.22 Because the NGFR reporter gene is of human origin, it provides advantages in terms of
increased specificity in SCID mouse models (compared with other mouse
reporter genes in use) and, presumably, reduced immunogenicity in
humans (compared with bacterial or mouse reporter genes). It was a
concern at the outset that endogenous NGFR expression in human
hematopoietic cells may complicate the SCID-hu bone assay. However, we
have not been able to detect cell-surface NGFR expression in primary
CD34+ cells, their progeny cells differentiated in vitro
and in vivo, or in fetal hematopoietic cells residing in the recipient
bone fragments in any of our experiments. In any case, although the presence of the full-length NGFR gene did not exert any noticeable adverse effects on the transduced hematopoietic precursors we evaluated, use of vectors like MINT expressing C-terminally truncated NGFR genes should alleviate any remaining misgivings. Therefore, the
stable and nonimmunogenic NGFR reporter coexpressed with a therapeutic
gene from the same vector may be useful in human gene therapy based on
human HSC as well as T cells,39 if an easily detectable
marker is desired to monitor and isolate transduced cells.
In summary, MSCV-based retroviral vectors encoding easily detectable
and selectable markers should facilitate studies aimed at further
characterizing the CD34+Lin subset
containing SRC. It is anticipated that the information gained will lead
to improvements in HSC-based gene and cellular therapies.
| |
FOOTNOTES |
Submitted December 3, 1997;
accepted February 20, 1998.
R.G.H. is supported in part by a grant from the National Cancer
Institute of Canada.
Address correspondence to Linzhao Cheng, PhD, Johns Hopkins University
School of Medicine and Osiris Therapeutics, Inc, 2001 Aliceanna St,
Baltimore, MD 21231; e-mail: LCheng{at}Osiristx.com.
Address reprint requests to Beth Hill, PhD, Systemix, Inc, 3155 Porter
Dr, Palo Alto, CA 94304.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Dr Richard Rigg for providing ProGag and ProPak-A
packaging cells, and to Dr Ivan Plavec for providing a LXSN-based
retroviral vector and an NGFR-containing plasmid. We also thank the
SyStemix Cell Processing Group for isolation of CD34+ cells
and the Comparative Medicine Group for production of SCID-hu mice.
| |
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[Abstract]
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B. Schiedlmeier, K. Kuhlcke, H. G. Eckert, C. Baum, W. J. Zeller, and S. Fruehauf
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice
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[Abstract]
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D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith
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Hematology,
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[Abstract]
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M. C. Dinauer, L. L. Li, H. Bjorgvinsdottir, C. Ding, and N. Pech
Long-Term Correction of Phagocyte NADPH Oxidase Activity by Retroviral-Mediated Gene Transfer in Murine X-Linked Chronic Granulomatous Disease
Blood,
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[Abstract]
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H. Miyoshi, K. A. Smith, D. E. Mosier, I. M. Verma, and B. E. Torbett
Transduction of Human CD34+ Cells That Mediate Long-Term Engraftment of NOD/SCID Mice by HIV Vectors
Science,
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[Abstract]
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Abstract
Introduction
Abstract