From the Departments of a Genetics, d Molecular Pharmacology, and e Microbiology and Immunology, Stanford University Medical School, Stanford, California 94305, the c Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, g Molecular Probes, Inc., Eugene, Oregon 97402, and the h Molecular Medicine Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98019
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ABSTRACT |
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Mutations in the acid The lysosomal storage disorders classified as
mucopolysaccharidoses (MPS)1
result from deficiencies in a number of enzymes responsible for the
catabolism of glycosaminoglycans. A heritable deficiency of the acid
hydrolase The basis for therapy in MPS VII and most other lysosomal storage
diseases is that secreted normal enzyme can be taken up into the
lysosomes of diseased cells, where it can degrade substrate (5). This
has been demonstrated in MPS VII mice by injection of recombinant
enzyme (6) and by transplantation of bone marrow (7, 8) or neural
progenitor cells (9). Somatic gene therapy using retrovirus
vector-mediated transduction of hematopoietic stem cells followed by
autologous transplantation of irradiated recipients showed that as
little as 2% of normal enzyme levels could completely reverse the
pathology in some major organs (10). Another strategy is autologous
implantation in vector-corrected fibroblasts in neo-organs (11). This
methodology has several technical advantages over stem cell
transduction, including ease of harvest of autologous target cells by
skin biopsy, ease of ex vivo expansion and retroviral
infection, and ease of removal of the neo-organ. Some of the clinical
manifestations of the disease, notably resolution of lysosomal storage
lesions in the liver and spleen, were ameliorated by continuous
intercellular exchange of the The efficacy of gene transfer has been limited, however, by the small
number of fibroblasts transplanted and by down-regulation of vector
expression (11, 12). One approach to overcoming these drawbacks may be
to increase the level of enzyme secreted from each cell to supply more
normal enzyme to the affected tissues (12, 13). Unfortunately,
enhancing Previously, we described a novel reporter assay utilizing E. coli Cells and Tissue Culture--
Murine MPS VII skin fibroblasts
(3521), fibroblasts isolated from a heterozygous mouse (3522) (13), and
primary cultures of BL/6 fibroblasts were grown in Dulbecco's modified
Eagle's medium supplemented with 10% (v/v) fetal calf serum, 50 nM 2-mercaptoethanol, 100 units/ml penicillin, 0.05 µM streptomycin, and 2.0 mM glutamine. 3521 fibroblasts were transduced with the retroviral expression vector
DCH Cell Lysate and Supernatant
Analyses--
Methylumbelliferyl-glucuronide (MUGlcu) (Molecular
Probes, Eugene, OR) fluorometric lysate assays were carried out
essentially as described (25). Briefly, adherent 3521 fibroblasts were
washed in phosphate-buffered saline and harvested by incubation in the presence of 0.2-0.5 ml of 0.25% trypsin-EDTA for 5 min at 37 °C. After washing in phosphate-buffered saline, cell suspensions were centrifuged at 400 × g, and the cell pellets were
resuspended by vigorous vortexing in 200 µl 0.2% (v/v) Triton X-100,
150 mM NaCl in H20 and frozen at Cell Staining, FACS Analysis, and Sorting--
Single cell
suspensions were pelleted and resuspended at a concentration of 4 × 106/ml in staining medium (biotin- and flavin-deficient
RPMI supplemented with 4% v/v FCS). Fifty µl of cells pre-incubated
for 10 min at 37 °C were mixed 1:1 (v/v) with 2 mM
fluorescein-di- Generation and Harvesting of Neo-organs--
The neo-organs were
prepared as described (11). Briefly, Gore-Tex fibers (a gift from
W. R. Gore, Inc., Taos, NM) were coated with collagen, sterilized by
UV light, and coated with 10 µg/ml recombinant fibroblast growth
factor-2 (Promega). The cells were harvested by trypsinization and
washed once with phosphate-buffered saline, and 5 × 106 cells/ml were resuspended in Dulbecco's modified
Eagle's medium containing 10% FBS, 5 mM Hepes, 100 µg
penicillin/streptomycin/fungizone, 1 µl (10 µg/ml) fibroblast
growth factor-2 (Promega), 0.25 µl (0.8 mg/ml) epidermal growth
factor (Boehringer Mannheim), and 0.5 ml (3 mg/ml) collagen in a dish
with evenly separated fibers. After incubation at 37 °C for 30 min
in the presence of 5% CO2, 2 volumes of medium were added,
and the incubation was continued for 2 days, by which time the
neo-organ contracted into a semisolid gel. The neo-organs were
transplanted subcutaneously through bilateral incisions on the back.
After 4 weeks, individual neo-organ were removed, single cell
suspensions were made by mincing and dispase digestion, and the explant
cultures were evaluated for enzyme expression.
FACS Analysis of DCH
Detection of Rare Cell Sorting Based on In Vivo Kinetics of Substrate Hydrolysis and Product
Retention--
We next wished to study in greater detail the kinetics
of substrate hydrolysis, and the retention of the product fluorescein, in viable cells. During the course of this work we generated another fluorescein based glucuronide substrate, PFBFDGlcu, which includes a
PFB moiety (Fig. 1A). We have established that PFB-based
substrates react with reactive protein thiols in vitro and
in vivo,2
enhancing intracellular retention compared with conventional fluorescein-based substrates (35). Mutant 3521 fibroblasts, wild-type
BL/6 fibroblasts, or a 1:1 mix of the two were loaded with FDGlcu or
PFBFDGlcu for various times and analyzed by FACS (Fig.
4A). Analysis of the mixed
samples reveals that both substrates yielded a bimodal fluorescence
distribution at all time points. However, with increasing incubation
time, resolution of the fluorescein dull (
In order to evaluate the relative capabilities of the two substrates to
resolve the negative and positive subpopulations, the 25th and 75th
percentiles were calculated for each mixed sample. These values
approximate the MFls of the 3521 and BL/6 subpopulations, respectively,
as there are equivalent numbers of each cell type present. By plotting
these values, as well as the MFls of 3521 and BL/6 samples stained
alone with each substrate, the extent of leakage of the hydrolysis
product from Heritable High Levels of Murine 3521 Fibroblasts Efficiently Endocytose
Target 3521 cells incubated in the presence of sorted DCH Isolation and Secondary Transplantation of Fibroblasts Stably
Expressing High Levels of
Although most silencing of vector expression occurs by 1-2 weeks after
transplantation (12), the remaining three primary transplant recipients
(six neo-organs) were kept for 2 months before analysis so the cells in
the primary and secondary grafts were maintained in vivo for
equivalent total lengths of time. FACS analysis of cells derived from
the six primary transplants revealed that a subpopulation of cells in
each neo-organ stably expressed The development of several quantitative FACS-based mammalian
reporter gene assays for bacterial hydrolytic enzymes (24, 31) prompted
us to test a similar approach for the detection of known deficiencies
in mammalian lysosomal hydrolases. The deficiency of Acid hydrolases, including Analysis of heterogeneous samples of An alternative method for detection of The efficacy of gene therapy for MPS VII depends on the level of enzyme
secreted by the corrected cells (10-13), and the FACS method that we
describe should be useful for isolating populations of cells producing
high levels of Although expression from Moloney-based proviral vectors is stable in
the majority of transduced fibroblasts after long term passage in
culture, even in the absence of selection, we and others have shown
that silencing occurs relatively rapidly after transplantation in the
mouse. However, in a small percentage of cells, expression can persist
even after long term passage in vivo (10, 12, 14, 15). We
applied the FACS assay to determine whether the population of
transduced fibroblasts expressing The secondary transplants derived from the G418-selected population had
higher percentages of long term stable expressing cells and
proportionally higher levels of Our results demonstrate that fibroblasts showing stable proviral
expression after passage in vivo can be isolated by cell sorting on the basis of PFBFDGlcu hydrolysis and have a higher probability of maintaining prolonged expression in vivo than
the original drug-selected population. The implications of these
results for gene therapy-based treatments are significant. In
vivo passage of transduced fibroblasts and perhaps other cell
types, followed by an ex vivo selection step, such as cell
sorting, may yield a subpopulation of cells harboring proviral
integrants with a significantly higher probability of stably expressing
the relevant therapeutic gene upon secondary transplantation.
Incorporating such a selection step may improve the clinical outcome of
somatic cell gene therapy-based approaches.
-glucuronidase gene lead
to systemic accumulation of undegraded glycosaminoglycans in lysosomes
and ultimately to clinical manifestations of mucopolysaccharidosis VII
(Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on
the levels of
-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of
enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the
quantitative analysis of
-glucuronidase activity in viable cells.
Murine mucopolysaccharidosis VII cells transduced with a
-glucuronidase retroviral vector can be isolated by cell sorting on
the basis of
-glucuronidase activity and cultured for further use.
In vitro analysis revealed that sorted cells have elevated levels of
-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing
-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can
be isolated by cell sorting and expanded ex vivo. A
relatively high percentage of these cells maintain stable expression
after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucuronidase (
-D-glucuronide
glucuronosohydrolase, EC 3.2.1.31) is responsible for the clinical
manifestations of MPS type VII (Sly disease) (1), which include
shortened life span, hepatosplenomegaly, skeletal deformities,
and mental retardation (2). Animal models share many of the
abnormalities found in human MPS VII (3, 4), including the
distention of lysosomes by granular and fibrillar storage material.
They have been used extensively as experimental systems to investigate pathogenesis and therapies.
-glucuronidase enzyme. The levels of
normal
-glucuronidase seen were similar to those that were effective
by hematopoietic stem cell transduction. However, there was no
improvement in macroscopic characteristics, such as skeletal
deformities. Similarly, enzyme secreted from fibroblast grafts in the
brain can correct storage lesions in neural cells near the graft
(12).
-glucuronidase activity by generating vectors with strong
or universal genetic regulatory elements may not be a viable solution,
as a number of enhancers gradually become inactivated in gene-corrected
fibroblasts in vivo (12, 14, 15). Recently, several
methodologies for the selection of retrovirus vector-transduced cells
have been described, utilizing bicistronic vectors that encode
transcriptional fusions between a therapeutic gene and a selectable
marker (16). Two classes of selectable marker genes have been used,
including those encoding proteins that confer resistance to otherwise
cytotoxic drugs, or surface markers that allow for selection by
fluorescence-activated cell sorting (FACS). However, a number of
problems associated with these surrogate markers have arisen, including
reduction in the translational efficiency of the downstream gene
(17-19), reduction in retroviral titers as a result of the excessive
size of the bicistronic DNA cassette (19), immunogenicity of the selectable marker (20), interference with normal cellular
differentiation (in the case of expression of an endogenous surface
protein) (21, 22), and repression of expression of the therapeutic gene
due to the presence of transcriptional silencer elements in prokaryotic drug resistance encoding genes (23). The complications associated with
the presence of surrogate markers might be avoided by selecting directly for expression of the therapeutic gene.
-glucuronidase in mammalian cells (24). In this report, we
describe the development of a FACS-based assay utilizing a fluorescein-based substrate for the detection of mammalian
-glucuronidase activity at the single cell level. This quantitative
assay can be used to isolate viable cells with uniformly high
-glucuronidase activity from a population of cells showing
heterogeneous levels of expression. Sorted cells can be expanded and
maintain high levels of endogenous and secreted
-glucuronidase in
culture. Utilizing the mouse model of MPS VII, this method was used to isolate a subpopulation of fibroblasts still expressing
-glucuronidase after in vivo subcutaneous passage in
neo-organs. Ex vivo expansion and secondary transplantation
of this subpopulation revealed that it was enriched for cells that
stably express high levels of
-glucuronidase in vivo. We
propose that this method may be a useful addition to retroviral-based
gene therapy treatment of MPS VII.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
H (DCH
H-3521) as described previously (13). The DCH
H
provirus encodes the human
-glucuronidase transcription unit 5' of
the retroviral enhancer in each long terminal repeat and the neomycin
resistance gene downstream of the 5' long terminal repeat. Where
indicated, transduced cells were selected by culturing in the presence
of 0.8 mg/ml G418 for 2 weeks. All cultures were maintained at 37 °C
in 5% CO2. Serum was heat-inactivated at 65 °C for 60 min to inactivate bovine
-glucuronidase. For cross-correction experiments, two-chamber, 24-mm plates with a 0.4 µm filter
(Transwell, Costar) separating the upper (donor cell) from the lower
(target cell) chamber were used, as described (25). Briefly, 1 × 105 3521 target cells resuspended in 2.5 ml of complete
media and an equivalent number of donor cells resuspended in 1.5 ml of
complete medium were added to the bottom and top wells of the cluster
plate, respectively. After 3.5 days in culture, supernatant and cells were harvested and assayed as described below.
70 °C.
Immediately prior to analysis, the cell homogenate was thawed,
vortexed, and centrifuged at 12,000 × g for 3 min to
remove cell debris. Fifty µl of supernatant harvested from the lower
target chamber was added to an equal volume of 10 mM MUGlcu
in 0.1 M citrate buffer (pH 4.5). The reactions were carried out at 37 °C for various times and stopped by the addition of 150 µl of 15 mM EDTA, 300 mM glycine (pH
11.2). For analysis of freshly sorted cells, 50 µl of 6 mM MUGlcu (in 0.1 M citrate buffer, pH 4.8, plus 0.3% Triton X-100) was added to 50 µl of sorted cells in
phosphate-buffered saline. For analysis of secreted enzyme, cell
supernatant was removed and centrifuged for 2 min at 12,000 × g to remove cell debris. MUGlcu (10 mM) in 0.1 M citrate buffer (pH 4.8) was added to the supernatant, and
the solution was incubated as described above. The fluorescent product,
4-methylumbelliferone, was measured on a 96-well plate with a
Fluoroskan II fluorescence plate reader (Flow Laboratories, McLean, VA)
(peak excitation at 355 nm, emission at 460 nm). A standard curve of
4-methylumbelliferone was assayed in parallel to calculate units (nmol
of methylumbelliferone/h) per mg of protein for the lysate assay or per
ml of supernatant. Cell protein content was measured in triplicate
using the DC protein assay (Bio-Rad). Cell pellets had 0.1-0.16 mg of
protein/24-mm well.
-D-glucuronide (FDGlcu) or
pentafluorobenzoylaminofluorescein-di-
-D-glucuronide (PFBFDGlcu) dissolved in staining medium (also equilibrated to 37 °C). After incubating for 10-90 min at 37 °C, the loading was stopped by dilution with 10 volumes of ice-cold staining medium. For
inhibition studies, 3521 fibroblasts were preincubated in 5 mM 1,4-saccharolactone (Sigma) or 0.3 mM
chloroquine at 37 °C for 10-30 min prior to the addition of
substrate. Cells were maintained in the presence of inhibitor
throughout the experiment. Cells were pelleted to remove any
fluorescent product in the medium and resuspended in 300 µl of fresh
staining medium supplemented with 1 µg/ml propidium iodide to
fluorescently stain dead cells. Cells were maintained on ice throughout
the FACS analyses to prevent leakage of the fluorescein product. FACS
set-up, analyses, and sorting were conducted as described (24, 26).
Histochemical staining with napthol AS-BI-
-D-glucuronide
(Sigma) was conducted as described (25).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
H-transduced 3521 Fibroblasts--
Mammalian
-glucuronidase shows broad specificity
for conjugated glucuronides, yielding D-glucuronic acid and
alcohol hydrolysis products. Although the fluorogenic substrate FDGlcu
(Fig. 1A) is efficiently
hydrolyzed by E. coli
-glucuronidase (24), specific activity of the mammalian enzyme against this substrate has yet to be
established. To determine whether FDGlcu is hydrolyzed by mammalian
-glucuronidase in viable cells,
-glucuronidase-negative 3521 fibroblasts or 3521 fibroblasts transduced with the
-glucuronidase double copy retrovirus vector DCH
H, which express high levels of
-glucuronidase (13), were incubated in the presence of FDGlcu and
analyzed by FACS. Viable DCH
H-3521 cells showed high levels of
fluorescein fluorescence (Fig. 1B). In contrast,
nontransduced 3521 cells incubated in the presence of FDGlcu are not
significantly more fluorescent than unstained controls. To
establish that the fluorescence detected results from
-glucuronidase specific hydrolysis of the substrate, 3521 cells were
incubated with the specific inhibitor 1,4-saccharolactone (27)
prior to the addition of FDGlcu (Fig. 1B). In the presence
of 1,4-saccharolactone, FDGlcu-loaded DCH
H-3521 cells showed
fluorescence levels comparable to cells incubated in the absence of
FDGlcu. These results show that
-glucuronidase is responsible for
the hydrolysis of FDGlcu, and that the fluorescent product can be
detected in viable cells by FACS.
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Fig. 1.
Structure and -glucuronidase-specific
hydrolysis of FDGlcu. The fluorogenic diglycoside FDGlcu
(A) includes two D-glucuronic acid residues
covalently bound via
linkages at the first carbon to fluorescein.
Addition of a pentafluorobenzoyl-amino group (shaded) yields
the substrate PFBFDGlcu, which shows similar spectral properties but
increased intracellular retention compared with FDGlcu (see under
"Results"). For either substrate, hydrolysis of both acetal
linkages (arrows) yields highly fluorescent fluorescein.
B, 3521 (
-glucuronidase-negative) and G418-selected
DCH
H-3521 (
-glucuronidase-positive) fibroblasts were preincubated
in the presence of 5 mM 1,4-saccharolactone or 300 µM chloroquine for 10 or 30 min, respectively, prior to
the addition of 1 mM FDGlcu. Samples incubated in the
absence of FDGlcu and inhibitor are also displayed. Viability was not
influenced by the presence of substrate or inhibitor (data not shown).
The MFl of each sample incubated in the absence of substrate is shown
in parentheses. The MFl for each sample incubated in the presence of
substrate is shown next to the relevant FACS histogram.
-Glucuronidase has optimal activity at pH 4.8, consistent with its
localization and degradative function in the acidic lysosome (28). To
determine whether the activity detected by FACS is primarily localized
in the endosomal/lysosomal compartment, 3521 and DCH
H-3521 cells
were incubated in the presence of the lysosomotropic weak base,
chloroquine, prior to the addition of substrate. The accumulation of
chloroquine leads to an increase in endosomal/lysosomal pH (29, 30),
inhibiting the activity of acid hydrolases in these organelles (31,
32). In the presence of chloroquine, fluorescence development of
FDGlcu-loaded DCH
H-3521 cells was significantly inhibited, to levels
comparable to those found in nontransduced cells or in DCH
H-3521
cells incubated in the absence of FDGlcu (Fig. 1B). These
results suggest that
-glucuronidase-mediated FDGlcu hydrolysis takes
place primarily in the endosomal/lysosomal compartment.
-Glucuronidase-positive Cells--
In
contrast to bulk lysate protocols, FACS-based assays measure
fluorescence on a single cell basis. As a result, very small populations of phenotypically distinct cells can be detected in a
heterogeneous population. To determine the sensitivity of the FACS-
-glucuronidase assay, 3521 and DCH
H-3521 cells were mixed at
ratios from 1:1 to 1000:1, loaded with FDGlcu, and analyzed by FACS
(Fig. 2A). For each mixed
population, the percentage of positive cells detected (G2) closely
matched the predicted value (Fig. 2B). Comparison of the
median fluorescence value (MFl) also allows for the quantitative
determination of the level of product associated with uncorrected
mutant cells. The MFl of 3521 cells (G1) decreased from 2.1 to 0.8 as
the 3521:DCH
H-3521 ratio increased from 1:1 to 100:1, and remained
at 0.8 as the ratio increased to 1000:1 (Fig. 2B). Although
some component of this difference is contributed by a concomitant
decrease in the number of DCH
H-3521 cells that fall in the G1 gate,
the MFl of 2.2 primarily reflects the level of product internalized by
the untreated mutant cells, and this figure is thus an overestimation
of the true MFl of the 3521 population. Nevertheless, even at a ratio
of 1:1, the majority of DCH
H-3521 cells show fluorescence values
distinct from those of 3521 cells, with a MFl 7-fold greater than the
latter. Most importantly, a population including 0.1% positive cells
can be accurately enumerated with this method.
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Fig. 2.
Detection of rare -glucuronidase-positive
cells by FACS. 250 to 2.5 × 105 G418-selected
DCH
H-3521 fibroblasts were mixed with 2.5 × 105
3521 fibroblasts and each population was incubated in the presence of 1 mM FDGlcu. A, 100,000 events were collected per
sample and displayed as 5% probability contour plots with outliers.
DCH
H-3521 and 3521 populations incubated in the absence of substrate
are also shown (gray plots). B, an electronic
gate in the fluorescein channel excluding all of the events collected
in the 3521 sample was applied to determine the percentages and MFls of
fluorescein-positive (G1) and -negative (G2) populations in each
sample. Autofluorescence was collected in the phycoerythrin
channel.
-Glucuronidase Activity/Substrate
Hydrolysis--
The broad distribution of fluorescence in
substrate-loaded DCH
H-3521 cells could result from heterogeneous
substrate loading or from cell-to-cell differences in the levels of
-glucuronidase expression. Given the influence of the integration
site (position effect variegation) on expression from the proviral long
terminal repeat (33, 34), heterogeneity in expression levels would be
expected from a population of cells comprised of a number of independent proviral integrants. To determine whether the fluorescence distribution reflects the intracellular
-glucuronidase expression level, DCH
H-3521 cells were loaded with FDGlcu, and viable cells were sorted on the basis of product fluorescence into lysis buffer for
analysis using the independent MUGlcu assay (Fig.
3A). Reanalysis of sorted
populations by FACS showed that the sorted fractions maintained their
relative fluorescence profiles (Fig. 3B). Lysate analysis
(Fig. 3D) of the bright sorted population (S2) showed levels
of activity approximately 2-fold higher than an equivalent number of
dull sorted cells (S1), a value consistent with that of the ratio of
the corrected MFls of these populations. Both S1 and S2 populations had
significantly higher levels of activity than nontransduced 3521 cells
when measured by FACS (Fig. 3C) and by lysate. Because dead
cells were excluded during the sort, and the S1 and S2 populations show
indistinguishable forward and orthogonal scatter profiles (data not
shown), the differences detected in
-glucuronidase activity were not
the result of differences in cell viability within the DCH
H-3521
population. Thus, the broad distribution of fluorescence detected in
the transduced population reflected the predicted heterogeneity in
expression levels.
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Fig. 3.
DCH H-3521 fibroblasts can be sorted on the
basis of
-glucuronidase activity. DCH
H-3521 or 3521 fibroblasts were incubated in the presence of 1 mM FDGlcu
as described in Fig. 1 and processed for cell sorting. A,
two sets of electronic gates were applied to sort the dullest (S1) and
brightest (S2) viable cells. B, a total of 30,000 viable
cells were sorted, per well, into staining medium and immediately
reanalyzed by FACS. C, viable 3521 fibroblasts were sorted
without gating in the fluorescein channel. D, alternatively,
cells were sorted in triplicate using the same electronic gates
directly into pH 4.5 lysate buffer and analyzed by lysate assay in the
presence of 3 mM MUGlcu. Mean units/well/30,000 cell
equivalents are shown for each sample. MFls for each sample are
displayed next to the relevant FACS histogram.
-glucuronidase-negative)
and bright (
-glucuronidase-positive) subpopulations is greater in
the presence of PFBFDGlcu. The superior sensitivity of the PFB-based
substrate is not due to an increase in substrate specificity, as the
fluorescence distributions of the
-glucuronidase-negative samples
stained with either substrate are indistinguishable. Thus, the
disparity must be attributed to a difference in the intracellular
retention of the fluorescent product in
-glucuronidase-positive
cells, which becomes apparent only in the presence of
-glucuronidase-negative cells.
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Fig. 4.
In vivo kinetics of FDGlcu versus
PFBFDGlcu hydrolysis and product retention. A,
-glucuronidase-negative 3521 cells (dashed line),
-glucuronidase+ BL/6 cells (solid line), or a
1:1 mix of the two (shaded histogram) were incubated in the
presence of FDGlcu or PFBFDGlcu for 10-90 min and analyzed by FACS.
For each time point, histograms of the homogeneous and mixed samples
are superimposed. The MFls of FDGlcu-loaded (open symbols)
or PFBFDGlcu-loaded (filled symbols) 3521 (squares in B) or BL/6 (squares in
C) samples were determined by calculating the 50th
percentile of the fluorescence data. For the 1:1 mix samples
(circles), the estimated MFls of the 3521 (B) and
BL/6 (C) subpopulations were determined by calculating the
25th and 75th percentiles of the fluorescence data values,
respectively. These values approximate the MFls of the 3521 and BL/6
subpopulations because the fluorescence distributions of the two are
nearly completely resolved.
-glucuronidase-positive cells and uptake by
-glucuronidase-negative cells can be determined. The MFl of the
negative subpopulation is clearly lower (Fig. 4B) and that
of the positive subpopulation higher (Fig. 4C) in the PFBFDGlcu-loaded samples. However, a low level of product leakage does
take place with PFBFDGlcu staining, as revealed by the reduced MFls of
the BL/6 subpopulation in the mixed sample compared with the BL/6
sample stained alone.
-Glucuronidase Expression, Secretion,
and Cross-correction by DCH
H-transduced Fibroblasts Sorted on the
Basis of
-Glucuronidase Activity--
We next wished to test
whether cells sorted on the basis of high
-glucuronidase enzymatic
activity show heritable levels of relatively high enzymatic activity,
and if so, whether these sorted cells secrete higher levels of
cross-correcting enzyme than the population from which they were
sorted. To generate a population of cells with relatively high levels
of
-glucuronidase activity, DCH
H-3521 fibroblasts were loaded
with PFBFDGlcu and gates were established such that the brightest 5%
of fluorescein-positive cells were sorted (data not shown). After 30 days in log-phase culture, the selected cells and the DCH
H-3521
population from which they were sorted were reanalyzed by FACS. The
sorted population maintained relatively high levels of
-glucuronidase activity, with 98% of the viable sorted cells
showing fluorescence levels greater than or equal to the brightest 4%
of viable DCH
H-3521 fibroblasts (Fig.
5A). The MFls of 0.49, 0.81, 2.4, and 79.0 for 3521, 3522, DCH
H-3521, and sorted DCH
H-3521
cells, respectively, were comparable with those obtained on the day of
sorting.
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Fig. 5.
Enhanced secretion of -glucuronidase and
cross-correction of
-glucuronidase deficient 3521 cells by
DCH
H-transduced fibroblasts sorted on the basis of high
-glucuronidase activity. A, 3521, 3522, DCH
H-3521, and DCH
H-3521 fibroblasts sorted on the brightest 5%
of cells (sorted DCH
H-3521) were reanalyzed by FACS 30 days post-sorting. Data for each population incubated in the presence
(light line) or absence (dark line) of PFBFDGlcu
prior to FACS analysis are displayed. To measure the level of
cross-correcting enzyme secreted by these cells, cultures of each were
established in parallel on Transwell dishes with target 3521 cells
plated in the lower chamber. Donor cells (B), supernatant
(C), and target cells (D) were harvested after
3.5 days in culture, and
-glucuronidase activity for each sample was
analyzed with the MUGlcu assay. All analyses were conducted in
triplicate, and the mean units (U) of activity per mg of
protein or per ml of medium (harvested from the lower chamber) are
shown for lysate and supernatant values, respectively. The S.E. is
displayed for each measurement.
-Glucuronidase--
Using these
-glucuronidase deficient cells
as targets for secreted enzyme, the cross-correcting enzyme in culture
supernatants can be accurately measured. Cultures of 3521, 3522, DCH
H-3521, and sorted DCH
H-3521 donor fibroblasts, were
established in Transwell plates that separated the donor from the
target cells by a fluid but not cell permeable membrane, precluding
enzyme transfer by cell-to-cell contact (25, 36). After 3.5 days in
culture, the donor cells, supernatants, and target cells were harvested and analyzed with the MUGlcu lysate assay and by FACS to compare the
cross-correcting activity generated by sorted cells with that generated
by the transduced, nonsorted population. Although enzymatic activity in
the target cells was not detectable using the FACS assay (data not
shown), lysate analysis revealed that the sorted population had a
2-fold greater level of intracellular
-glucuronidase activity than
the cells from which they were sorted (Fig. 5B), demonstrating that the FACS assay can be used to enrich for cells expressing high levels of
-glucuronidase. Both the DCH
H-3521 and
the sorted DCH
H-3521 populations showed significantly higher levels
of expression than 3522 cells, which express normal levels of
-glucuronidase (13). The relative levels of
-glucuronidase secreted by each population was consistent with the donor cell lysate
analysis (Fig. 5C). Enzymatic activity in the culture
supernatant of the sorted DCH
H-3521 population was approximately
2-fold greater than that detected for the DCH
H-3521 population from
which it was sorted and 20-fold greater than that detected for 3522 cells.
H-3521
cells showed about 2-fold greater enzymatic activity than 3521 cells
incubated in the presence of the unsorted transduced population (Fig.
5D), results that are consistent with enzyme levels detected
in the supernatant. As expected, no activity was detected in target
cells incubated in the presence of 3521 donor cells. The linear
relationship between the level of
-glucuronidase in the supernatant
and that endocytosed by
-glucuronidase deficient cells shows that
saturation of the mannose-6-phosphate endocytic machinery (37) did not
occur under the culture conditions used. Thus, the FACS can be used to
select for cells with very high levels of
-glucuronidase expression
and secretion, from a population already expressing a high level of
-glucuronidase.
-Glucuronidase after in Vivo Passage in
the MPS VII Mouse--
Rapid silencing of retroviral expression after
transplantation of genetically modified fibroblasts is well documented
(12, 14, 15). However, a subpopulation of transplanted cells showing stable expression even after long term passage in vivo can
be detected (11, 12, 15). The presence of rare expressing cells may
reflect a stochastic process of silencing, whereby all proviruses have
an equal probability of silencing over time. Alternatively, such cells
may represent clones in which the proviral integration site supports
long term expression. If the latter explanation is correct, then the
long term expressing population should show greater stability of
expression upon secondary transplantation than the original
transplanted population. To test this hypothesis, an in vivo
experiment was conducted as described in Fig.
6. Neo-organs (11, 38) of G418 resistant
DCH
H-3521 cells (80.0% FACS-positive, not shown) were transplanted
into four adult MPS VII mice. The neo-organs from one mouse were
removed 1 month after transplantation, and the cells were explanted.
Cells from one of the neo-organs (5786R, 6.4% positive as measured by
FACS, Table I) were used to generate new
neo-organs for secondary transplantation. Half of the cells were loaded
with the PFBFDGlcu substrate, and
-glucuronidase-positive cells were
isolated by FACS. Because both vector-expressing and cross-corrected
cells may be positive by this method, the other half of the explant
culture was G418-selected to isolate only those cells expressing the
vector. Neo-organoids were prepared from the sorted and from the
G418-selected populations and transplanted as secondary grafts into MPS
VII mice.
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Fig. 6.
Scheme for testing stability of vector
expression by PFBFDGlcu FACS selection after in vivo
passage. Neo-organs (see under "Experimental
Procedures") of DCH H-3521 cells were transplanted subcutaneously
into adult MPS VII mice. One month later, the neo-organs from one mouse
were surgically removed, and the cells were explanted and analyzed for
-glucuronidase expression. Cells from one neo-organ (6.4%
-glucuronidase-positive by FACS) were used for secondary
transplants. Half of the cells were subjected to FACS (PFBFDGlcu
substrate) to isolate
-glucuronidase-positive cells, and half were
G418-selected to isolate only the cells expressing the vector. The
cells were expanded, and new neo-organs were prepared from both
populations for secondary transplantation into MPS VII mice. To analyze
the cells for the same total length of time in vivo, the
remaining primary grafts were removed 2 months after transplantation,
and the secondary grafts were removed after 1 month. Explant cultures
were established from each neo-organ and the cells were analyzed for
total enzyme activity and proportion of positive cells.
-Glucuronidase expression in DCH
H retrovirus vector-corrected
fibroblasts after primary and secondary transplantation in neo-organs
H-3521 cells were transplanted into adult MPS VII
mice according the scheme shown in Fig. 6. The cells derived from the
neo-organs after primary and secondary transplantation were analyzed
for quantitative enzymatic activity and for proportion of positive
cells, by two methods. The explanted primary cells were maintained in
culture for a total of about 1 month before secondary transplantation
(32 days for selected cells; 35 days for sorted cells), which included
the times needed to establish the explant cultures, select or sort the
cells, and expand the cultures to obtain enough cells to construct the
secondary neo-organs. ND, not determined; 2°, secondary.
-glucuronidase at percentages
ranging from 0.7 to 20.2% with a mean of 5.2% (Table I). This degree
of silencing in vivo was similar to our previous
observations in the MPS VII mouse (10,
12).3 The secondary
transplanted neo-organs were removed after 1 month and analyzed for
-glucuronidase expression (Table I). The secondary transplants
derived from both the sorted and G418-selected populations had
significantly increased percentages of
-glucuronidase-positive cells
(17.2 and 44.8%, respectively) compared with the primary group
(5.2%). The FACS analysis was verified by counting samples of the
explanted cells stained by a histochemical reaction for
-glucuronidase enzymatic activity (39). Calculation of the mean
percentage of positive cells for each transplant group revealed no
significant differences between the two methods (Table I). The amount
of
-glucuronidase enzymatic activity was generally proportional to
the percentage of positive cells in individual neo-organs (Table
I).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucuronidase
causing MPS VII is an excellent test system because both mouse and
large animal models are available, and they have been used extensively
for enzyme replacement, transplantation, and gene therapy experiments.
Furthermore, analysis of mammalian
-glucuronidase with a number of
potential substrates has shown that this enzyme will hydrolyze almost
any aglycone in a
-linkage to glucuronic acid (40). Here, we show
that fluorescein-
-D-glucuronide substrates are
internalized and hydrolyzed by human or murine
-glucuronidase in
viable cells at levels sufficient for detection and isolation by FACS.
-glucuronidase, are present in an acidic
pre-lysosomal compartment of the endocytic pathway (41, 42), and the
endosome is the primary site of initial exposure of endocytosed
material to hydrolytic enzymes (43). Analysis of FDGlcu-loaded
fibroblasts by confocal fluorescence microscopy revealed a
punctate cytosolic staining pattern indicative of endosomal/lysosomal localization (data not shown). Thus, our results are most consistent with a model of substrate internalization involving fluid phase pinocytosis with subsequent hydrolysis of the endocytosed substrate in
acidified endosomes and/or lysosomes. Consequently, the FACS assay
described here may have greater physiological relevance than the lysate
assay, which measures the entire cellular content of
-glucuronidase.
However, as endocytic capacity and the size of the lysosomal
compartment is dependent on the cell type (44) and state of activation
(45), the fluorescence intensities detected under distinct culture
conditions or in different cell types may not accurately reflect
relative levels of intracellular
-glucuronidase. Furthermore,
determination of the number of
-glucuronidase molecules within a
cell using this methodology is confounded by several variables inherent
to ex vivo analysis, including sequestration of the enzyme
in compartments inaccessible to the endocytosed substrate,
heterogeneity in autofluorescence levels, and heterogeneity in the pH
of endocytic compartments, which strongly influences fluorescein
fluorescence (29).
-glucuronidase-positive and
-negative fibroblasts allowed for direct comparison of the loading and
retention properties of the substrates PFBFDGlcu and FDGlcu. The MFl
ratio of
-glucuronidase-positive to -negative cells was
significantly greater with the PFBFDGlcu substrate than with FDGlcu,
indicating that the signal to background ratio is superior for the
former. The pentafluorobenzoyl moiety is covalently bound in
vitro and in vivo to reactive thiols, such as
glutathione.2 Such a covalently modified substrate would be
more hydrophilic and thus less likely to cross the plasma membrane,
which could explain the superior retention properties of PFBFDGlcu.
Furthermore, the hydrolysis product pentafluorobenzoylaminofluorescein
has a significantly lower pKa than that of
fluorescein (pKa = 6.0 versus 6.7, respectively). As a result, a higher fraction of the former is
fluorescent at the relatively low pH of the endosomal/lysosomal compartment, thus giving a stronger signal per molecule of substrate cleaved. In cells with low
-glucuronidase expression, the levels of
intracellular fluorescein generated may be insufficient for detection
by FACS. Although
-glucuronidase activity at 0 °C is very low,
the enzyme shows a reduction in activity of only 9-10-fold at 15 °C
versus 37 °C (Q10 = 3.7) (data not shown). Thus, the sensitivity of the assay can be increased by shifting the incubation to
15 °C, instead of 0 °C, allowing for further hydrolysis while preventing product efflux, which is inversely related to temperature (47).
-glucuronidase by flow
cytometry, using monoclonal antibodies specific for the enzyme, was
reported previously (48, 49). However, this method requires fixation
and permeabilization of the cells, which precludes using it for
enrichment of viable gene-corrected cells. Furthermore, the presence of
cross-reactive material in MPS VII patients (50) clearly deficient in
lysosomal enzymatic activity also limits the use of antibody staining,
which measures the level of
-glucuronidase protein (including
mutant protein) present, rather than the physiologically relevant
enzymatic activity level. Another approach is to co-express cell surface proteins, as surrogate markers, with the therapeutic gene
from a vector (51, 52). However, several potential complications may
preclude clinical use of such markers, including immunogenicity of the
encoded protein (20) and interference with normal cellular differentiation (21, 22). Clearly, avoiding the requirement for
surrogate markers by direct selection for expression of the functional
therapeutic gene, is a desirable goal. Detection of enzymatic activity
at the single cell level, and the potential for detecting very rare
cells, makes this FACS assay superior to other methods for the
detection of genetically corrected cells. Given the availability of a
number of fluorescein-based hydrolase substrates, this assay may be
applicable to the detection of other exoglycosidases responsible for
lysosomal storage disorders, as we have shown with
-glucocerebrosidase-deficient Gaucher disease cells (35).
-glucuronidase prior to autologous transplantation.
Because the amount of
-glucuronidase secreted by fibroblasts
transduced with
-glucuronidase expression vectors is proportional to
cellular expression levels (13, 25), we chose to test whether cells
sorted on the basis of high
-glucuronidase expression produced
higher levels of cross-correcting enzyme than the parental population
from which they were sorted. Although most of the cells in the unsorted
population were positive for DCH
H (data not shown), the broad
heritable distribution of expression, attributable to position-effects
(33, 34), allowed for the isolation by FACS of a subset of cells that
stably expressed the highest levels of
-glucuronidase.
-glucuronidase after primary
transplantation was resistant to silencing upon secondary
transplantation. Neo-organs were used to simplify the retrieval of
transplanted cells (11), which could then be sorted on the basis of
-glucuronidase expression and expanded ex vivo prior to
secondary transplantation. Because the DCH
H vector also encodes the
neomycin resistance gene, cells could also be selected on the basis of
G418 resistance. Our results clearly demonstrate that in
vivo passage followed by selection (either sorting or drug
administration) yields a subpopulation of cells with a significantly higher probability of maintaining expression upon transplantation than
the original population of transduced cells. We hypothesize that this
population is composed of clones in which the proviral integration
sites support long term expression of the therapeutic gene, and primary
transplantation essentially serves as an in vivo selection
step. Determining what factors are involved in retroviral silencing in
the in vivo milieu would be of particular use in the
development of ex vivo selection protocols.
-glucuronidase activity compared
with the secondary transplants of sorted cells. We hypothesize that the
sorted population included both transduced and cross-corrected cells,
whereas the drug-selected population contained only vector-transduced cells. This was supported by the observation that the sorted primary cells used for the secondary neo-organ were 88.0% positive by cytochemical stain (94.4% by FACS) but decreased to 49.9% positive after several passages in vitro (data not shown). Because
transduced cells show stable expression after in vitro
passage (Fig. 5 and Refs. 13, 25, and 39), the cells that became
negative were probably cross-corrected rather than vector-transduced.
This is concordant with our previous experiments that showed that
cross-corrected MPS VII cells rapidly lose
-glucuronidase activity
when a high concentration source of donor enzyme is removed, as a
result of normal proteolytic degradation of the endocytosed protein in
the lysosome (25). The neomycin phosphotransferase protein is not transferred between cells and thus may provide a more stringent means
of selection, but the disadvantages associated with the inclusion of a
drug-selectable marker make the sorting approach more expedient for
clinical applications. Furthermore, cells expressing the vector at low
levels may be selected against by G418. This would be a disadvantage
where it is desirable to transplant as many positive cells as possible,
such as with hematopoietic stem cells (10, 46). The sorting method can
be used to select the transduced cells from an infected population
where no selectable marker is included in the vector design
(35)3 and may thus result in better overall delivery of the
therapeutic protein in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. Richard Haugland (Molecular Probes, Eugene, OR) for helpful suggestions and for supplying the substrates described in this manuscript. We are grateful to Drs. David Parks, Peter Katsikis, and William Kerr for advice and help throughout the course of this project.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants CA42509 (to L. A. H.); AI35304 (to G. P. N.); and DK42707, DK46637, and DK54481 (to J. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Supported by National Institutes of Health Training Grant GM07790. Present address: Molecular Medicine Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98019.
f G.P.N. is a Scholar of the Leukemia Society of America and a recipient of the Burrough's Wellcome New Investigator Award in Pharmacology.
i To whom correspondence should be addressed: Dept. of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-2324; Fax: 215-573-5952; E-mail: jhwolfe{at}vet.upenn.edu.
The abbreviations used are:
MPS, mucopolysaccharidosis; MFl, median fluorescence value; FACS, fluorescence-activated cell sorting; MUGlcu, methylumbelliferyl-glucuronide; FDGlcu, fluorescein-di--D-glucuronide; PFB, pentafluorobenzoylamino.
2 Z. Diwa, unpublished observations.
3 J. H. Wolfe, unpublished observations.
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REFERENCES |
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