|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 829-836
GENE THERAPY
Long-term multilineage expression in peripheral blood from a
Moloney murine leukemia virus vector after serial transplantation of
transduced bone marrow cells
Timothy W. Austin,
Suzan Salimi,
Gabor Veres,
Franck Morel,
Heini Ilves,
Roland Scollay, and
Ivan Plavec
From SyStemix Inc, Palo Alto, CA.
 |
Abstract |
Using a mouse bone marrow transplantation model, the authors
evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in
vivo tracking of transduced cells. NGFR+ cells were
detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood
erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells
of all mice for up to 8 months. Transgene expression in individual mice
was not blocked in the various cell lineages of the peripheral blood
and spleen, in several stages of T-cell maturation in the thymus, or in
the Lin /loSca-1+ and
c-kit+Sca-1+ subsets of bone
marrow cells highly enriched for long-term
multilineage-reconstituting activity. Serial transplantation of
purified NGFR+c-kit+Sca-1+
bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted
mice. The authors concluded that MMLV-based vectors were capable of
efficient and sustained transgene expression in multiple lineages of
peripheral blood cells and hematopoietic organs and in hematopoietic
stem cell (HSC) populations. Differentiation of engrafting HSC to
peripheral blood cells is not necessarily associated with dramatic
suppression of retroviral gene expression. In light of earlier studies
showing that vector elements other than the long-terminal repeat
enhancer, promoter, and primer binding site can have an impact on
long-term transgene expression, these findings accentuate the
importance of empirically testing retroviral vectors to determine
lasting in vivo expression.
(Blood. 2000;95:829-836)
© 2000 by The American Society of Hematology.
| |
Introduction |
Hematopoietic stem cells (HSC) are key targets for gene
therapy approaches to treat inherited hematologic disorders, cancers, and chronic viral diseases such as acquired immunodeficiency syndrome. Successful application of stem cell-based gene therapies to treat blood
system disorders will require efficient delivery of the therapeutic
gene to engrafting HSC, long-term reconstitution of hematopoiesis from
transduced HSC, and stable expression of the therapeutic gene in the
affected blood cell lineages. Most clinical trials using retroviral
vectors to transfer therapeutic genes into long-term
multilineage-reconstituting HSC have relied on derivatives of the
Moloney murine leukemia virus (MMLV). Although retroviral marking is
routinely observed at high levels in mice reconstituted with transduced
HSC, marking is low in clinical trials and in primate models.
Additionally, stable expression of therapeutic genes from retroviral
vectors remains problematic even in mouse bone marrow transplantation models.
The MMLV long-terminal repeat (LTR) promoter can direct gene expression
in blood, spleen, and thymus cells after the transplantation of
transduced bone marrow cells to lethally irradiated
mice,1,2 and it is active in multiple hematopoietic cell
lineages of reconstituted mice, including T cells, B cells, myeloid
cells, and erythroid cells.3,4 However, MMLV LTR-driven
gene expression generally declines as a function of time after
transplantation, and it varies between individual mice and within
various tissues analyzed from a single mouse. Moreover, transgene
expression from MMLV-based vectors can be drastically reduced by 70%
to more than 90% in secondary or tertiary colony-forming units-spleen
(CFU-S) derived from serial transplantation of transduced bone marrow
cells.5,6
Although the ultimate reasons for the decline and variability in
transgene expression after transplantation are unclear, several studies
have suggested that the MMLV LTR is transcriptionally silenced in
hematopoietic stem/progenitor cells, their progeny, or both after
transplantation.5-7 Indeed, the lack of expression from
retroviral vectors in CFU-S after serial transplantation was correlated
with increased methylation of the LTR.5,6 However, in
previous studies RNA levels or enzymatic activity were used to measure
expression of the therapeutic gene from cell lysates. Consequently, it
is unclear whether the MMLV LTR promoter is active at uniform levels in
each cell of the hematopoietic tissues or whether expression varies
from cell to cell and between cell types within each tissue.
Understanding which is the more accurate picture for retroviral vector
expression in HSC and their progeny after transplantation may enable a
rational approach toward improved long-term retroviral vector
expression. Analysis of MMLV LTR promoter-driven transgene expression
at the single cell level would help to clarify whether transgene
expression is restricted in HSC, its progeny cells, or both.
For several years we have been developing a stem cell gene therapy
approach to deliver genes that blocks replication of the human
immunodeficiency virus (HIV) in hematopoietic cells of infected persons.8,9 Success with this approach requires gene
transfer to multilineage-reconstituting HSC and long-term expression of anti-HIV genes throughout the hematopoietic system, particularly in
progeny cells targeted by HIV, such as CD4+ T cells and
macrophages. In this study, we evaluated an MMLV-based vector bearing a
combination of 2 anti-HIV genes for long-term expression in a mouse
bone marrow transplantation model. Expression of a cell-surface marker
gene included in the vector (the human nerve growth factor receptor
[NGFR10]) was tracked at the single cell level by flow
cytometry as a measure of MMLV LTR promoter activity. We showed that a
high frequency of transgene-positive cells and high levels of vector
expression can be achieved in multiple lineages of peripheral blood
cells for 8 months after transplantation. Transgene expression was
evident in all cell lineages examined from the peripheral blood and
spleen, in successive stages of T-cell differentiation in the thymus, or in highly enriched bone marrow stem/progenitor cell populations. In
addition, we demonstrated that retroviral gene expression is maintained
after engraftment and subsequent differentiation of HSC during
peripheral blood reconstitution after transplantation in secondary recipients.
| |
Materials and methods |
Retroviral vectors and producer cell lines
Construction and anti-HIV efficacy of the 1171 vector will be
described elsewhere (Veres G, manuscript in preparation). The 1171 vector encoded the RevM1011 and the HIV-1 pol antisense genes cloned in pLN.9 The truncated NGFR10
served as a marker to trace expression in vivo. Ecotropic retroviral
vector producer cells were derived from the GP-E86 packaging cell
line.12
Retrovirus transduction of bone marrow cells
Eight- to 12-week-old
(C57BL/Ka.AKR/J)Sys-Ptprca-Thy-1a mice (BA .1;
Thy-1.1, Ly-5.2) were used as bone marrow donors. Eight- to 12-week-old
congenic (C57BL/6J.SJL/J)Sys-Ptprcb-Thy-1b mice
(B6SJL; Thy-1.2, Ly-5.1) were the recipients in our bone marrow
transplantation experiments. Mice were bred and maintained at the
SyStemix animal facility. B.A1 mice were injected with 5-fluorouracil
(150 mg/kg body weight) 5 to 6 days before bone marrow harvest. Cells
were flushed from femoral shafts with phosphate-buffered saline (PBS)
and 0.2% bovine serum albumin (BSA) and were plated at
106/mL in Whitlock/Witte medium containing 10% fetal
bovine serum (FBS; Hyclone Laboratory, Logan, UT), murine stem cell
factor (100 ng/mL), murine IL-3 (10 ng/mL), and murine IL-6 (10 ng/mL). Cytokines were from R&D Systems (Minneapolis, MN). Cells were cultured
for 24 hours at 37°C, plated onto irradiated (1500 rad) virus
producer cells, and co-cultured for an additional 48 hours with
protamine sulfate (16 µg/mL) added to the media. Nonadherent bone
marrow cells were recovered, resuspended in PBS/0.2% BSA, and
106 cells (200µL) was injected through the tail vein into
lethally irradiated (2 × 525 rad) B6SJL recipient mice.
Analysis of transgene expression on peripheral blood cells,
thymocytes, and splenocytes
Peripheral blood samples were collected from individual mice by
retro-orbital bleeding and analyzed independently. A portion of the
sample was stained with a phycoerythrin (PE)-conjugated antibody
specific for human NGFR (Boehringer Mannheim, Indianapolis, IN) and a
fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD41 antibody
(Pharmingen, San Diego, CA) and analyzed for the NGFR expression on
erythrocytes and platelets. Red blood cells (RBCs) were defined using
characteristic forward/side-scatter properties, and platelets were
defined by forward/side-scatter properties and CD41 expression. Both
populations were clearly distinguished from leukocytes, which comprised
less than 0.5% of the events. The remainder of the sample was diluted
in hypotonic lysis buffer to remove erythrocytes, and the remaining
cells were stained with anti-Ly-5.2 (CD45.2)-FITC (Pharmingen),
anti-NGFR-PE, and a lineage-specific allophycocyanin (APC)-conjugated
antibody (either Gr-1, Mac-1 [CD11b], CD3e, or B220 [CD45R], all
from Pharmingen). After the final wash, cells were resuspended in
PBS/2% FBS with propidium iodide (1 µg/mL), and dead cells were
excluded from analysis based on propidium iodide staining and
forward-scatter properties. The thymus and spleen from individual mice
8 months after transplantation were made into single-cell suspensions
and stained with anti-Ly-5.2-FITC and anti-NGFR-PE. Splenocytes were additionally stained with APC-conjugated anti-Gr-1, anti-Mac-1, anti-B220, anti-CD4, or anti-CD8 (Pharmingen). Thymocytes were stained
either with anti-CD8-FITC (Pharmingen), anti-NGFR-PE, and anti-CD4-APC,
or with anti-Ly-5.2-FITC and anti-NGFR-PE. Analysis was performed on a
FACScalibur instrument (Becton Dickinson Immunochemistry Systems, San
Jose, CA). The significance of differences in transgene expression in
peripheral blood cells was analyzed using a 1-tailed Student's
t test.
Analysis and purification of transgene expressing HSC
populations
Total bone marrow cells were collected from the long bones (2 femurs
and 2 tibias) of each mouse by flushing with PBS/0.2% BSA. To analyze
NGFR expression on Lin/loSca-1+ cells,
bone marrow cells from mice 8 months after transplantation were
depleted of RBCs by hypotonic lysis and were stained with anti-Ly-5.2-FITC, anti-NGFR-PE, anti-Sca-1 biotin, APC-conjugated antibodies to lineage-specific antigens (CD4, CD8, Gr-1, Mac-1, and
B220), and streptavidin-Texas red. NGFR expression was analyzed on
Lin/loSca-1+ cells from whole bone
marrow or from lineage antigen-depleted cells prepared by removal of
lineage antigen-stained cells by immunomagnetic bead depletion (Dynal,
Great Neck, NJ). Analysis of bone marrow HSC populations from mice 8 months after transplantation with the phenotype
c-kit+Thy-1.1loLin/loSca-1+
was performed as previously described.13 Briefly, cells
were incubated with a biotinylated anti-Sca-1 antibody and enriched using the MACS system for magnetic bead selection (Miltenyi Biotech, Auburn, CA). Sca-1 enriched cells from individual mice were stained with anti-Thy-1.1-FITC, a cocktail of PE-conjugated antibodies specific
for lineage markers, anti-c-kit-APC, and streptavidin-Texas red, and
analyzed on a FACStarplus instrument (Becton Dickinson
Immunochemistry Systems). For sorting of donor-type
NGFR+c-kit+Sca-1+ HSC
populations, Sca-1 enriched cells were pooled from 2 mice, stained with
anti-NGFR-PE, anti-c-kit-APC, and streptavidin-Texas red,
resuspended after the final wash in PBS/2% FBS with propidium iodide,
and sorted on a FACStarplus instrument (Becton Dickinson
Immunochemistry Systems). Sorted cells were resuspended in PBS/0.2%
BSA and injected through the retro-orbital sinus into lethally
irradiated (2 × 525 rad) B6SJL recipient mice.
DNA analysis
High molecular weight genomic DNA was isolated from spleens of
individual mice 8 months after transplantation by proteinase K/sodium
dodecyl sulfate/RNAase extraction.14 DNA recovered after
phenol extraction was ethanol precipitated and resuspended in TE
buffer. Proviral integration pattern was analyzed by Southern blot
analysis after digestion of 10µg genomic DNA with Hind III, which
cleaves the vector 15 nucleotides 5' to the predicted NGFR translational start site. Proviral copy number was analyzed by digestion with Hind III and Bcl I, which released a 2-kb fragment containing the NGFR and pol/AS sequences. Southern blots were probed
with a 1.9-kb fragment (Hind III/Hind II) spanning the NGFR and pol/AS
sequences. The probe was generated by the random priming method using
[32P]dCTP.
The proviral integrant copy number was quantitated relative to that of
the single-copy interferon-° gene (using a 1.2-kb Pst I/Hind III genomic DNA fragment as a probe15) by
densitometry using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager system.
| |
Results |
Long-term transgene expression on peripheral blood cells
The MMLV-based retroviral vector 1171 (L-M10-IRES-NGFR-pol/AS)
encodes 3 genes from 1 polycistronic mRNA transcript: the RevM10 gene,11 the truncated form of the NGFR,10 and
an antisense fragment from the HIV-1 pol gene (pol/AS; see Figure
4D).9 Translation of the NGFR protein is mediated by the
internal ribosomal entry site (IRES) of the human encephalomyocarditis
virus16 and thus is linked to the RevM10-pol/AS expression.
The expression of RevM10 and a cell surface marker protein (whose
translation is mediated by the IRES) are co-linear8,17;
therefore, the more easily detectable NGFR surface marker was used to
facilitate the tracking of retroviral gene expression by flow
cytometry. Ecotropic retroviral vector producer cells were derived from
the GP-E86 packaging cell line.12 Transduction of
5-fluorouracil bone marrow and reconstitution of lethally irradiated
mice were carried out in 2 independent experiments. In each experiment, groups of 7 to 8 mice (male and female) received either 1171 or mock-transduced bone marrow cells. After 5 months, 4 mice were killed
and evaluated for any abnormalities associated with long-term retroviral vector transgene expression in gene-modified cells. Of the
remaining mice, 3 from experiment 2 were killed 8 months after
transplantation and analyzed in detail for transgene expression in
various hematopoietic organs (splenocyte and thymocyte subsets and
c-kit+Sca-1+ bone marrow cells). Four
mice were analyzed for transgene expression in
Lin/loSca-1+ bone marrow cells. Four
mice were killed, and
NGFR+c-kit+Sca-1+ bone
marrow cells were isolated by cell sorting and were used in secondary
transplantation experiments. Chimerism of donor cells was monitored by
Ly-5.2 expression in various hematopoietic tissues. Overall, no
striking differences were evident in the kinetics of hematopoietic
recovery, the extent of donor-type reconstitution of peripheral blood
cells, or the proportions of various peripheral blood cell types
between the groups of mice receiving 1171 or mock-transduced
cells.29
Four to 5 weeks after transplantation, NGFR+ cells were
detected in the circulation of all transplanted animals. The frequency of NGFR+ cells ranged from 16.3% to 44.7% for white blood
cells, 2.7% to 25.6% for platelets, and 23.6% to 38.7% for
erythrocytes. NGFR expression was repeatedly detected in multiple
peripheral blood lineages (RBCs, platelets, and donor-type WBCs) for
more than 8 months in all mice analyzed (Figures
1 and 2). In
experiment 1, the percentage of NGFR+ RBCs, platelets, and
donor WBCs declined with time and then did not significantly decline
further after the second month (Figure 2). Similar results were
obtained in experiment 2 for RBCs and donor WBCs, though the percentage
of NGFR+ platelets was lowest at day 38 and stabilized at a
higher level after the second month. Overall, the frequency of
NGFR+ RBCs, platelets, or donor WBCs declined 1- to
2.5-fold by 8 months after transplantation (in experiment 2, NGFR+ platelets increased 2.7-fold overall).
View larger version (58K):
[in this window]
[in a new window]
| Fig 1.
Flow cytometric analysis of NGFR transgene expression in
peripheral blood cells on day 249 after transplantation.
Peripheral blood cells were prepared as described in the "Materials
and Methods" from a representative mouse (217). (top left) Region 3 (R3) denotes the NGFR+ fraction of red blood cells (RBCs).
(top right) R2 is the Ly-5.2+ donor type fraction of live
gated white blood cells (WBCs). WBC subsets are donor-type myeloid
(Gr-1+ or Mac-1+), B (B220+), and T
(CD3e+) cells gated on live cells and R2. PLTs, platelets.
The percentage of NGFR+ cells in each is shown in Table
1.
|
|
View larger version (23K):
[in this window]
[in a new window]
| Fig 2.
Sustained multilineage transgene expression from an
MMLV-based vector in peripheral blood for more than 8 months after
transplantation of transduced bone marrow cells.
Data represented the mean percentage ± SEM of NGFR+
cells in peripheral blood of mice at various time-points after
transplantation of bone marrow cells transduced with the 1171 vector.
Results are shown from 2 independent transductions, experiment 1 (top)
and experiment 2 (bottom). NGFR expression was analyzed as in Figure 1
with WBCs gated on live donor-type cells. Controls are nonirradiated,
age-matched mice from the donor and recipient strains, mocks are mice
transplanted with nontransduced cells otherwise prepared as for 1171 transduced cells. Eight mice were analyzed in experiment 1; 7 mice were
analyzed in experiment 2 until day 161, and 3 were analyzed on day 249. Note that overall NGFR expression in RBCs, platelets, and donor WBCs
declined with time then stabilized after the second month. In
experiment 1, the percentage of NGFR+ cells was greater at
day 31 than at any subsequent time-point (P < .03, except
for donor WBCs at day 31 versus day 89). Similar results were obtained
in experiment 2 (P < .05), except that fewer
NGFR+ platelets were detected at day 38 than at later
time-points. After the second month after transplantation, no other
significant declines (P < .05) in the percentage of
NGFR+ WBCs, RBCs, or platelets were observed between any of
the time-points in either experiment. All other abbreviations are as in
Figure 1.
|
|
NGFR was detected in all peripheral donor WBC subsets analyzed,
including B (CD45R B220+), T (CD3e+), and
myeloid (Gr-1+ or CD11b Mac-1+) cells in each
mouse (Table 1 and Figures 1 and
3). NGFR expression was variable between
mice and between different phenotypic populations of peripheral blood
cells. Unexpectedly, the percentage of NGFR+ cells was
significantly greater in donor type Gr-1+ or
Mac-1+ myeloid cells than in RBCs, platelets, or donor
lymphoid cells (B220+ or CD3e+) at all
time-points (Figure 3). The only exception to this observation was for
Mac-1+ versus B220+ cells in mouse 213 at day
249 (Table 1). When both experiments were combined, the frequency of
NGFR+ cells was greater in
Ly-5.2+Gr-1+ cells than in
Ly-5.2+B220+ or
Ly-5.2+CD3e+ cells, RBCs, or platelets at all
time-points in 13 of 15 mice. These data clearly show that MMLV
LTR-driven transgene expression is not blocked in any of the
predominant peripheral blood lineages in long-term reconstituted mice.
Significantly, because at day 249 NGFR expression was at background
levels on host-type WBC subsets (ie, 0.1% to 0.4% of
Ly5.2 cells positive for Gr-1, Mac-1, B220, or CD3e
were NGFR+), we ruled out the possibility that transgene
expression was sustained from the continuous in vivo transfer of the
vector or NGFR protein from injected transduced cells to nontransduced
cells. It is possible that the greater frequency of NGFR+
myeloid cells resulted in part from the lineage-specific enhancement of
LTR or IRES activity.
View this table:
[in this window]
[in a new window]
|
Table 1.
Enduring multilineage hematopoiesis reconstituted from
transgene-expressing cells after transplantation with transduced bone
marrow
|
|
View larger version (22K):
[in this window]
[in a new window]
| Fig 3.
Stable long-term transgene expression in all peripheral
donor white blood cell types with preferential representation of
NGFR+ cells in Gr-1+ and Mac-1+
donor WBCs.
NGFR expression was analyzed by flow cytometry as in Figure 1. Data
represented the mean percentage ± SEM of NGFR+ cells in
peripheral blood of 7 mice from experiment 2 at various time-points
after transplantation of bone marrow cells transduced with the 1171 vector. There were no significant declines (P < .05) in the
percentage of NGFR+ cells within a given phenotypic cell
population sampled after the second month after transplantation. Note
that the frequency of NGFR+ cells was consistently greater
in Ly5.2+Gr-1+ or
Ly5.2+Mac-1+ myeloid cells than in all other
cell types at each time-point. For Gr-1+ versus
B220+, CD3e+, RBCs, or platelets,
P < .001 (n = 7) at days 116 and 161, and
P < 0.03 (n = 3) at day 249. For Mac-1+
versus B220+, CD3e+, RBCs, or platelets,
P < .009 (n = 7) at days 116 and 161, and
P < .02 (n = 3) at day 249, except for Mac-1+
versus B220+. All abbreviations are as in Figures 1 and
2.
|
|
Persistent retroviral gene expression in hematopoietic organs
Long-term retroviral transgene expression was further demonstrated
in various hematopoietic organs of 3 mice analyzed 8 months after
transplantation. In the thymus, more than 80% of total thymocytes were
Ly-5.2+. CD4/CD8 ratios in the thymi of transplanted mice
(average, 4.1; n = 4) were not significantly different from those of
the age-matched control mice (3.5 and 3.7). On average,
28% of Ly-5.2+ thymocytes expressed the NGFR, and
expression varied between mice and between different phenotypic
populations of the thymus. However, expression was detected in all mice
examined on each of the Ly-5.2+ phenotypic thymocyte
subsets, CD4CD8,
CD4+CD8+, CD4+, and
CD8+ cells (Table 2). Hence, it
was not restricted at any stage of thymocyte maturation studied. In
mouse 213, CD4+CD8+ cells comprised less than
1% of total thymocytes, thereby precluding analysis of NGFR expression
in this compartment.
In the spleen, more than 91% of cells were donor Ly-5.2+,
and overall 23% of Ly-5.2+ splenocytes expressed the NGFR
(Table 3). Again, expression varied from
mouse to mouse and between different phenotypic populations. Each
Ly-5.2+ lineage subset expressed the NGFR, including
Gr-1+ or Mac-1+ myeloid cells, CD4+
or CD8+ T cells, and B220+ B cells. Of the
various Ly-5.2+ splenocyte populations, NGFR expression was
consistently detected on more Gr-1+ myeloid cells than on
the other phenotypes. When phenotypic subsets from spleen were compared
to peripheral blood of the same mouse, NGFR expression varied though
the trend was for expression on a greater proportion of donor
Gr-1+ or Mac-1+ cells in peripheral blood than
in the spleen. NGFR expression was also variable when compared to donor
CD4+ or CD8+ cells in the spleen versus the
thymus.
Southern blot analysis was carried out to assess the gene marking
frequency and the number of proviral integrants in the spleens of
representative mice with long-term transgene expression 8 months after
transplantation. We calculated that 0.4, 1.2, and 2.5 proviral copies
were detected per genome from mouse 211, 213, and 217, respectively
(Figure 4); 4 to 6 proviral integrants were
detected in the splenocytes of each mouse. Thus, the NGFR+
splenocytes of each mouse were derived from 4 to 6 or fewer
retrovirally marked clones.
View larger version (45K):
[in this window]
[in a new window]
| Fig 4.
Southern blot analysis of integration pattern and
proviral copy number in splenocytes of mice with long-term multilineage
transgene expression.
DNA was isolated from splenocytes of mice whose transgene expression is
shown in Table 3 and was digested with Hind III (A, lanes 1-4) or Hind
III and Bcl I (B,C, lanes 1-4). (A,B) Southern blot was probed with a
1.9-kb Hind III/Hind II vector fragment encoding the NGFR and pol/AS
cDNA. (C) The same blot was reprobed with a genomic DNA fragment from
the interferon- gene. The blot was exposed to autoradiographic film
for 4 days in all mice except mouse 211, which was exposed for 8 days.
(D) Schematic diagram of the retroviral vector and NGFR pol/AS DNA
probe used in A and B.
|
|
Long-term transgene expression in bone marrow HSC populations
Overall, all blood cell lineages of each mouse examined expressed
the NGFR, regardless of whether cells were from peripheral blood or
hematopoietic organs. The finding that the NGFR was detected for 8 months on Ly-5.2+Gr-1+ peripheral blood cells
suggests continuous replenishment of short-lived granulocytes
(half-life, approximately 24 hours)18 by hematopoietic precursors expressing the retroviral transgene. Additionally, retroviral gene expression was detected throughout the various stages
of T-cell development in the thymus and in peripheral blood T cells.
Based on these data, it is likely that retroviral gene expression is
sustained in long-term multilineage-reconstituting cells. This
possibility was evaluated in
Lin/loSca-1+ and in
c-kit+Sca-1+ bone marrow cells; both
populations are highly enriched for long-term multilineage-reconstituting cells in normal19,20 or
reconstituted mice.21 Total or Lin+-depleted
Lin/loSca-1+ bone marrow cells from mice
8 months after transplantation were analyzed for Ly-5.2 and NGFR
expression. NGFR expression ranged from 0.6% to 22.2% on
Lin/loSca-1+ cells (Table
4).
Several studies have shown that the
Lin/loSca-1+ cells are a heterogeneous
population of long-term and transiently reconstituting multipotent
cells.20,22,23 Expression of the c-kit gene is a
key marker of long-term reconstituting cells, and
Lin/loSca-1+c-kit
cells lack detectable long-term reconstituting
potential.20,24,25 Retroviral gene expression was further
examined in c-kit+Sca-1+ cells enriched
from bone marrow of individual mice by positive selection for
Sca-1+ cells. As shown in Figure
5, total bone marrow or
Sca-1+-enriched cells expressing relatively high levels of
the c-kit+ and Sca-1+ antigens are a
distinct population on dual-parameter dot plots. Stringent gating of
this c-kit+Sca-1+ population showed
that more than 96% of these cells were Ly-5.2+ and more
than 94% were Lin/lo in 3 reconstituted mice 8 months after transplantation and in age-matched control mice (data not
shown). Using this gating strategy, 20% to 22% of
Lin/loSca-1+ cells were
c-kit+, and
Thy-1.1loLin/lo cells comprised 42% to
53% of c-kit+Sca-1+ cells from the 3 reconstituted mice or control mice. NGFR expression in the
c-kit+Sca-1+ population was limited to
the Ly-5.2+ donor-type cells and was distributed evenly
between the Thy-1.1 and Thy-1.1lo cells
(data not shown). In addition to these 3 mice, bone marrow cells were
pooled in 2 independent experiments from 2 mice 8 months after
transplantation. Overall, 6.7% to 22.7% of
c-kit+Sca-1+ cells from all long-term
reconstituted mice analyzed were NGFR+ (Table 4). The mean
fluorescence intensity of NGFR staining was reduced in
Lin/loSca-1+ or
c-kit+Sca-1+ cells relative to more
mature bone marrow or peripheral blood cells. Consistently fewer
NGFR+ cells were detected in the HSC populations than in
the donor WBC of each mouse.
View larger version (33K):
[in this window]
[in a new window]
| Fig 5.
FACs analysis of transgene expression in bone marrow
c-kit+Sca-1+ stem/progenitor cells 8 months after transplantation.
Bone marrow from 4 long bones of each mouse was recovered, Sca-1
selected, and analyzed by flow cytometry as described in "Materials
and Methods." (left) c-kit versus Sca-1 staining of
live-gated cells. (boxes) Regions used to define
c-kit+Sca-1+ cells. (right) Histograms
of NGFR expression on live-gated
c-kit+Sca-1+ cells. (markers)
Proportion of NGFR+ cells. Two age-matched control mice
(BA.1 and B6SJL) and 3 mice transplanted with bone marrow cells
transduced with the 1171 vector are shown.
|
|
Transgene expression in peripheral blood of secondary transplant
recipients
The potential of phenotypically defined bone marrow populations for
multilineage reconstitution and continued retroviral gene expression
was evaluated in 2 experiments. In each experiment, NGFR+c-kit+Sca-1+ cells
were purified from pooled bone marrow of 2 mice 8 months after
transplantation by cell sorting and were transferred to lethally
irradiated recipients. In the first experiment, recipient mice received
approximately 1900 NGFR+c-kit+Sca-1+ cells,
and, after 2 months, the RBCs, platelets, and donor WBCs were
reconstituted in 5 of 5 mice with Ly-5.2+NGFR+
cells. Two mice received 200,000 total bone marrow cells pooled from
these donors for comparison with the HSC-enriched grafts. Injection of
approximately 100 NGFR+c-kit+Sca-1+ cells in
the second experiment resulted in the multilineage reconstitution of
Ly-5.2+NGFR+ cells in 3 of 5 mice. Donor
reconstitution of sorted cells measured 2 months after transplantation
ranged from 12% to 52%, and NGFR expression was detected on 13% to
60% of Ly-5.2+ WBCs, 4% to 40% of RBCs, and 4% to 35%
of platelets (Table 5). The mean percentage
NGFR+ cells in the peripheral blood of the secondary
transplant recipient mice was approximately twice that of the primary
recipients measured at 2 to 3 months after transplantation. The
variance in NGFR expression (SEM) was greater in the secondary
transplant recipients, perhaps because fewer mice were used here than
in the primary transplant groups.
View this table:
[in this window]
[in a new window]
|
Table 5.
Multilineage NGFR expression on peripheral blood cells
after secondary BMT with sorted NGFR+
c-kit+ Sca-1+ cells
|
|
| |
Discussion |
In this study, we analyzed the potential of the MMLV promoter to
direct long-term transgene expression in the peripheral blood, hematopoietic organs, and bone marrow stem/progenitor compartment of
reconstituted mice. FACS analysis of peripheral blood showed that
multilineage reconstitution with NGFR+ donor-type cells
early (4 to 5 weeks) after transplantation was sustained for up to 8 months in all the mice studied from 2 independent transduction
experiments. Similar data were obtained in 2 experiments with related
vectors in which the RevM10 translation start site was abolished
(unpublished results). Careful hematologic and histologic analyses did
not reveal any abnormalities associated with long-term transgene
expression in gene-modified cells.29
Previous studies have shown that MMLV-based vectors are expressed in
the peripheral blood, spleen, thymus, or bone marrow after
transplantation.1-4 However, transgene expression levels generally declined with time and varied between individual mice and
between different tissues of the mouse. Although we observed an overall
decline in transgene expression during the first 2 months after
transplantation, long-term expression beyond the second month was
remarkably stable in all lineages examined. Several experimental
differences in the current study might have contributed favorably to
our observation of consistent, long-term, multilineage transgene
expression. We evaluated transgene expression frequency on a per cell
basis in donor-derived cells; many earlier studies scored transgene
expression from cell lysates. The NGFR marker gene used here had not
been, to our knowledge, evaluated previously in the mouse bone marrow
transplantation setting. It is possible that the combination of
detection reagents and expression level at the cell surface results in
a greater detection frequency for transgene-expressing cells. It is
also possible that the immunogenicity or negative influence on
hematopoiesis of this gene product is less pronounced or negligible
than other marker genes (ie, CD24, CD8, mPrP, or the neomycin
phosphotransferase gene).2 Although the level of
therapeutic transgene expression in target cells required for clinical
efficacy varies according to each disease indication, the data
presented here show that stable, long-term transgene expression can be
achieved from MMLV-based retroviral vectors in a variety of blood cell
lineages found in the periphery or in hematopoietic organs.
The decline in retroviral vector expression after transplantation of
transduced bone marrow cells has been attributed to restricted expression in long-term multilineage-reconstituting HSC and their progeny compared to transient multilineage-reconstituting cells or more
mature cells.5,6 Support for this proposal comes from the
finding that serial transfer of bone marrow cells can lead to
undetectable MMLV-LTR-driven transgene expression in 70% to 90% of
secondary or tertiary CFU-S.5,26 Decreased expression over
time has also been observed with vectors having LTRs and additional
elements from other murine retroviruses.27,4 However, the
issue of how transgene expression in phenotypically or functionally defined HSC populations relates to that in peripheral blood cells remains controversial. Analysis of a murine Friend spleen
focus-forming virus vector showed that though transgene expression was
detected in bone marrow Lin/loSca-1+
stem/progenitor cells 2 to 4 months after transplantation, expression declined in WBCs from 100% at 4 weeks to 23% of mice positive at week
28.28 In contrast, preselection of cells expressing a CD24
transgene from a murine stem cell-based vector resulted in high-level,
long-term transgene expression (81%) in bone marrow Lin/loSca-1+ cells after
transplantation, yet only 28% of peripheral WBCs expressed the
transgene.7 Clearly, the observation of a greater frequency
of transgene expression in bone marrow stem/progenitor cells than in
peripheral blood is at odds with the view that retroviral gene
expression is suppressed in HSC but not in more mature blood cells.
We demonstrated that NGFR expression was sustained 8 months after
transplantation in a significant fraction of bone marrow Lin/loSca-1+ stem/progenitor cells, and
in at least some of the primitive c-kit+Sca-1+ subset enriched for
long-term multilineage-reconstituting HSC. Transgene expression was
consistently detected in fewer
Lin/loSca-1+ or
c-kit+Sca-1+ bone marrow cells than in
peripheral WBCs. However, long-term expression was stable in peripheral
blood cells and was clearly demonstrated in multiple lineages of the
peripheral blood and spleen cells 8 months after transplantation.
Moreover, transgene expression was detected throughout the various
stages of T-cell lineage differentiation from the bone marrow
stem/progenitor compartment and immature
CD4+CD8+ thymocytes to the more mature
CD4+ or CD8+ thymocytes and peripheral
CD3e+ T cells. Additionally, NGFR was expressed in splenic
and peripheral B220+ B cells. In the peripheral blood,
granulocytes have a relatively short half-life (approximately 24 hours)18 and are continually replenished from progenitor
cells in the bone marrow. The consistent detection of the NGFR on donor
type Gr-1+ cells for up to 8 months suggested continuous
production of NGFR+ granulocytes from NGFR+
progenitors. Similarly, sustained production of NGFR+
platelets was likely supported by NGFR+
megakaryocytes in the bone marrow.
Transplantation of
NGFR+c-kit+Sca-1+ cells
into secondary recipients demonstrated that these HSC populations are
sufficient for hematopoietic recovery and multilineage reconstitution
of lethally irradiated mice. Significantly, all secondary recipients
were repopulated with NGFR+ RBCs, platelets, and donor-type
WBCs, directly demonstrated for the first time that retroviral gene
expression in multilineage-reconstituting HSC was sustained in the
peripheral blood progeny cells after serial transplantation. The serial
transplantation data, in conjunction with the analysis of primary
transplant recipients, provided strong evidence that silencing of MMLV
LTR-driven transgene expression is most pronounced in long-term,
multilineage-reconstituting HSC during the hematopoietic recovery phase
after myeloablation and transplantation. Once durable chimerism of
transgene-expressing HSC is established, progeny cells expressing the
transgene are continuously generated and contribute to the various
peripheral blood cell lineages.
Other investigators5,6 have reported that pronounced
silencing of MMLV expression at the mRNA level is accompanied by extensive methylation of the viral LTR in secondary CFU-S. Although long-term expression in the peripheral blood of secondary recipients was not studied, it is likely that the MMLV vector would be silenced there as well. Multiple modifications of the vector LTR and primer binding site were shown to improve the frequency of transgene expression from proviral integrants.6 In our study,
relatively few retrovirally marked clones contributed to spleen
reconstitution (4 to 6 or fewer). Because the proviral copy number per
cell was greater than the percentage of NGFR+ cells, it is
likely that not all proviral integrants were transcriptionally active.
Nonetheless, the finding that transgene expression persisted for at
least 8 months demonstrated that some of the proviral integrants were
not silenced. Because vector elements other than the LTR enhancer,
promoter, and primer binding site can have an impact on long-term
transgene expression (eg, addition of the neomycin phosphotransferase
gene),2 our findings emphasized the critical role
for in vivo analysis when evaluating the potential of
retroviral vectors for long-term expression in the
hematopoietic system.
| |
Acknowledgments |
The authors thank Nobuko Uchida and Stan Tamaki for help with KTLS cell isolation.
| |
Footnotes |
Submitted November 18, 1998; accepted September 28, 1999.
Reprints: Timothy W. Austin, SyStemix Inc, 3155 Porter Drive,
Palo Alto, CA 94304; email: tim.austin{at}pharma.novartis.com.
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.
| |
References |
1.
Correll PH, Fink JK, Brady RO, Perry LK, Karlsson S.
Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells.
Proc Natl Acad Sci U S A.
1989;86:8912[Abstract/Free Full Text].
2.
Apperley JF, Luskey BD, Williams DA.
Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression.
Blood.
1991;78:310[Abstract/Free Full Text].
3.
Moore KA, Scarpa M, Kooyer S, Utter A, Caskey CT, Belmont JW.
Evaluation of lymphoid-specific enhancer addition or substitution in a basic retrovirus vector.
Hum Gene Ther.
1991;2:307[Medline]
[Order article via Infotrieve].
4.
Rivière I, Brose K, Mulligan RC.
Effect of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.
Proc Natl Acad Sci U S A.
1995;92:6733[Abstract/Free Full Text].
5.
Challita PM, Kohn DB.
Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
Proc Natl Acad Sci U S A.
1994;91:2567[Abstract/Free Full Text].
6.
Robbins PB, Skelton DC, Yu XJ, Halene S, Leonard EH, Kohn DB.
Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells.
Proc Natl Acad Sci U S A.
1998;95:10,182[Abstract/Free Full Text].
7.
Pawliuk R, Eaves CJ, Humphries RK.
Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen.
Hum Gene Ther.
1997;8:1595[Medline]
[Order article via Infotrieve].
8.
Bonyhadi M, Moss K, Voytovitch A, et al.
RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.
J Virol.
1997;71:4707[Abstract].
9.
Veres G, Junker U, Baker J, et al.
Comparative analyses of intracellularly expressed antisense RNA as inhibitors of human immunodeficiency virus type 1 replication.
J Virol.
1998;72:1894[Abstract/Free Full Text].
10.
Mavilio F, Ferrari G, Rossini S, et al.
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood.
1994;83:1988[Abstract/Free Full Text].
11.
Malim MH, Freimuth WW, Liu J, et al.
Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication.
J Exp Med.
1992;176:1197[Abstract/Free Full Text].
12.
Markowitz D, Goff S, Bank A.
A safe packaging line for gene transfer: separating viral genes on two different plasmids.
J Virol.
1988;62:1120[Abstract/Free Full Text].
13.
Uchida N, Friera AM, He D, Reitsma MJ, Tsukamoto AS, Weissman IL.
Hydroxyurea can be used to increase mouse c-kit+Thy-1.1loLin-/lo Sca-1+ hematopoietic cell number and frequency in cell cycle in vivo.
Blood.
1997;90:4354[Abstract/Free Full Text].
14.
Maniatis T, Fritsch EF, Sambrook J.
Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.
15.
Plavec I, Papayannopoulou T, Maury C, Meyer F.
A human  -globin gene fused to the human -globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells.
Blood.
1993;81:1384[Abstract/Free Full Text].
16.
Jang SK, Davies MV, Kaufman RJ, Wimmer E.
Initiation of protein synthesis by internal entry of ribosomes into 5' nontranslated region of encephalomyocarditis virus RNA in vivo.
J Virol.
1989;63:1651[Abstract/Free Full Text].
17.
Su L, Lee R, Bonyhadi M, et al.
Hematopoietic stem cell-based gene therapy for acquired immunodeficiency syndrome: efficient transduction and expression of RevM10 in myeloid cells in vivo and in vitro.
Blood.
1997;89:2283[Abstract/Free Full Text].
18.
Boggs DR, Winkelstein A.
White Cell Manual. 4th ed. Philadelphia: Davis; 1983.
19.
Spangrude GJ, Heimfeld S, Weissman IL.
Purification and characterization of mouse hematopoietic stem cells.
Science.
1988;241:58[Abstract/Free Full Text].
20.
Morrison SJ, Weissman IL.
The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.
Immunity.
1994;1:661[Medline]
[Order article via Infotrieve].
21.
Morrison SJ, Wandycz AM, Hemmati HD, Wright DE, Weissman IL.
Identification of a lineage of multipotent hematopoietic progenitors.
Development.
1997;124:1929[Abstract].
22.
Spangrude GJ, Johnson GR.
Resting and activated subsets of mouse multipotent hematopoietic stem cells.
Proc Natl Acad Sci U S A.
1990;7:7433.
23.
Smith LG, Weissman IL, Heimfeld S.
Clonal analysis of hematopoietic stem-cell differentiation in vivo.
Proc Natl Acad Sci U S A.
1991;8:2788.
24.
Ikuta K, Weissman IL.
Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.
Proc Natl Acad Sci U S A.
1992;89:1502[Abstract/Free Full Text].
25.
Orlic D, Fischer R, Nishikawa S, Nienhuis AW, Bodine DM.
Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor.
Blood.
1993;82:762[Abstract/Free Full Text].
26.
Moore KA, Fletcher FA, Villalon DK, Utter AE, Belmont JW.
Human adenosine deaminase expression in mice.
Blood.
1990;75:2085[Abstract/Free Full Text].
27.
Correll PH, Colilla S, Karlsson S.
Retroviral vector design for long-term expression in murine hematopoietic cells |
Top
Abstract
Introduction