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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 363-369
Protein Replacement by Receptor-Mediated Endocytosis Corrects the
Sensitivity of Fanconi Anemia Group C Cells to Mitomycin C
By
Hagop Youssoufian,
Frank A.E. Kruyt, and
Xiaotong Li
From the Department of Molecular and Human Genetics and the
Department of Medicine, Baylor College of Medicine, Houston, TX.
 |
ABSTRACT |
Current methods for direct gene transfer into hematopoietic cells
are inefficient. Here we show that functional complementation of
Fanconi anemia (FA) group C cells by protein replacement can be as
efficacious as by transfection with wild-type FAC cDNA. We expressed a
chimeric protein (called His-ILFAC) consisting of the mature coding
portion of gibbon interleukin-3 (IL-3) and full-length FAC in
Escherichia coli. The purified bacterial protein is
internalized by hematopoietic cells via IL-3 receptors. The intracellular half-life of His-ILFAC is approximately 60 minutes, which
is comparable to that of the transgene-encoded FAC protein. In this
cell-culture model His-ILFAC completely corrects the sensitivity of FA
group C cells to mitomycin C, but it has no effect on FA cells that
belong to complementation groups A and B. We suggest that
receptor-mediated endocytosis of cytokine-fusion proteins may be of
general use to deliver macromolecules into hematopoietic progenitor
cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
FANCONI ANEMIA (FA) is widely regarded as
a prototype hematopoietic disorder that is amenable to gene
therapy.1,2 FA is genetically heterogeneous and manifested
clinically by birth defects, progressive failure of hematopoiesis, and
transformation to acute leukemia.3 Over the past several
years, great strides have been made in the genetic classification of FA
into distinct complementation groups, in the assignment of chromosomal
loci for three groups, and in cloning of the disease genes for
complementation groups A and C, called FAA and FAC,
respectively.4,5 The latter two groups account for the
majority of FA patients. Although the FAA and FAC genes
encode unique proteins with no homology to each other,6-8
all FA cells are classically distinguished by having an enhanced
sensitivity to bifunctional cross-linking agents, such as mitomycin C
(MMC) and diepoxybutane (DEB). In addition, FA cells have a propensity
to apoptosis and manifest variable cell cycle defects.9,10
Progress has also been made toward an understanding of the function of
proteins encoded by these genes. The FAC protein has been shown to
localize predominantly to the cytoplasmic compartment11-16
and function in a prerepair pathway.13 Indeed, cytoplasmic
localization of FAC is essential for cross-linker (MMC and DEB)
complementation of FA group C cells, whereas forced entry into nuclei
completely abolishes this function.13 FAC also binds to
NADPH cytochrome P450 reductase and attenuates its activity, which
suggests that it plays a role in cellular detoxification.17
By contrast, FAA localizes to both the cytoplasm and
nucleus,15,18,19 and forced exit from the nucleus abolishes its cross-linker complementation function.19
Death in FA usually results from complications of hematopoietic
failure.2 Although many questions still remain about the molecular pathogenesis of FA, parallel efforts to improve the therapy
of this devastating disorder by bone marrow and cord blood transplantation are yielding encouraging results. Gene therapy is also
being explored, although to date the success of this approach has been
limited.20 The central premise of gene therapy is to introduce genes directly into target cells of interest. The paucity of
hematopoietic stem or progenitor cells and other unknown barriers for
successful transduction and expression may be difficult to overcome
with current methods. Here we show an alternative method for functional
complementation of hematopoietic cells using cell culture models of FA
group C. This method exploits the ability of a cytokine ligand-receptor
complex to undergo endocytosis. We show that a cytokine fusion protein
can be internalized by a similar mechanism and correct a deficient
function of FA cells.
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MATERIALS AND METHODS |
Cell culture and transfection.
Lymphoblasts and WEHI cells were maintained in RPMI 1640 medium
containing 10% fetal calf serum (FCS). Two different human lymphoblastoid cell lines were used in these studies: HSC536 (compound heterozygous for a missense mutation in FAC on one allele and an unknown mutation on the second allele) and GM4510 (homozygous for a
splicing mutation in FAC). HSC72 (FA group A) and HSC230 (FA
group B) cells have been described previously.6 Stably transfected lymphoblasts generated by electroporation were grown in
RPMI 1640-10% FCS supplemented with 200 µg/mL hygromycin B.
Constructs.
A 1.66-kb BamHI-Xba I restriction fragment containing
full-length human FAC was obtained from pGEX2TK-FAC, as described
previously,21 and cloned into the corresponding restriction
sites of pQE9 (QIAGEN, Valencia, CA) to obtain pQE9-FAC. This construct
would be expected to encode a His-tagged full-length FAC protein. To
derive pQE9-ILFAC, a 0.4-kb interleukin-3 (IL-3) cassette was obtained
by polymerase chain reaction (PCR) using the primers
5 -CGGGATCCGCTCCCATGACCCAGACAA-3 and
5-CGGGATCCAGAGATCTCAAGGCTCAAAGT-3, which contain
artificial BamHI sites. After amplification of the mature
coding region of gibbon IL-3 from pXM-IL-3 (gift of Dr Steve Clark,
Genetics Institute),22 the BamHI-digested PCR
fragment was inserted into the unique BamHI site of pQE9-FAC.
The sequence and reading frame of the junctions were confirmed by DNA
sequencing. This arrangement added two amino acids, Gly-Ser, between
the carboxy terminus of IL-3 and amino terminus of FAC. The
His-IL-3-FAC cDNA insert was also subcloned into the BamHI and
Xba I sites of the mammalian episomal expression vector pDR2
downstream of an ATG in the context of a Kozak consensus sequence (details of this construction are available upon
request).18
Expression and purification of His-tagged proteins.
The plasmids pQE9-FAC and pQE9-ILFAC were used to transform
Escherichia coli strain M15 (pREP4). A 50-mL culture of LB
containing ampicillin and kanamycin bacteria grown overnight at
37°C was expanded to 1-L culture medium, grown to
A595 = 0.7, induced with 1 mmol/L
isopropyl- -D-thiogalactopyranoside and grown for an additional 4 hours at 37°C. Bacteria were pelleted by centrifugation (5,000g, 10 minutes) and resuspended (5 g wet weight into 35 mL) in Buffer I (0.1 mol/L NaH2PO4, pH 8.0, and
10 mmol/L Tris-HCl, pH 8.0, and protease inhibitors). Using a French
Press, the resuspended bacteria were lysed twice and crude inclusion
bodies pelleted by centrifugation (10,000g, 15 minutes).
Inclusion bodies were resuspended in Buffer I supplemented with 1 mol/L
guanidine-HCl and 0.2% NP40, and then repelleted by centrifugation.
Washed inclusion bodies were dissolved at room temperature in Buffer A
(Buffer I containing 6 mol/L guanidine-HCl, 0.2% NP40, and 10 mmol/L
2-mercaptoethanol). The supernatant was clarified twice by
centrifugation (10,000g, 15 minutes) and incubated with
Ni2+-agarose (0.5 mL of 50% suspension in Buffer A per 15 mL lysate) for 1 hour at room temperature. The slurry was loaded onto
PolyPrep columns (Bio-Rad Laboratories, Richmond, CA) and washed
successively with 50 bead volumes of Buffer B (0.1 mol/L
NaH2PO4, 10 mmol/L Tris-HCl, 8 mol/L urea [pH
8.0], 0.2% NP40, and protease inhibitors), Buffer C (same as Buffer
B, except for pH 6.3), and Buffer D (same as Buffer B, except for pH
5.9 and absence of NP40). Soluble fusion protein was obtained by
elution with 10 mL of Buffer E (0.1 mol/L NaH2PO4, 10 mmol/L Tris-HCl, 8 mol/L urea [pH
4.5]). After stepwise dialysis against phosphate-buffered saline (PBS;
pH 7.5) containing 4 mol/L urea, 2 mol/L urea, and no urea. The
His-ILFAC protein was purified further by fast protein liquid
chromatography on a Superdex-75 column (Pharmacia Biotech Inc,
Piscataway, NJ). Purified recombinant protein was concentrated by
centrifugation through a CentriPrep-10 column (Amicon, Beverly, MA).
Binding and internalization studies.
Purified His-ILFAC or His-FAC were added to HSC536 cells at 4°C in
RPMI 1640-10% FCS. After 10 minutes, cells were centrifuged, fresh
medium was added in the absence of His-tagged proteins, and cells were
rapidly warmed to 37°C. Internalization and intracellular turnover
of the proteins were assessed by washing the cells rapidly with cold
PBS, lysis in 1× Laemmli buffer, and analysis for the presence of
FAC-related proteins by Western blotting.
Western blotting.
The affinity-purified anti-FAC antibody and the Western blotting
procedure have been described previously.10,12,14 Briefly, cells were lysed in a buffer containing 20 mmol/L Tris-HCl, pH 8.0, 50 mmol/L NaCl, 2 mmol/L EDTA, 0.1% NP40 and supplemented with 0.5 mmol/L
phenylmethylsulfonyl fluoride (PMSF). Aliquots representing 1 × 105 HSC536 cells or column fractions from the gel
chromatography step were subjected to electrophoresis on a 10%
polyacrylamide gel (SDS-PAGE) and transferred to Polyscreen membrane
(NEN Life Science Products, Boston, MA). Blots were incubated with
affinity-purified FAC antibody in 10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.05% Tween 20, and 5% nonfat dry milk, followed by
incubation in the same buffer with peroxidase-conjugated
goat-anti-rabbit IgG (GIBCO-BRL, Grand Island, NY). Bands were detected
by using enhanced chemiluminescence (Amersham Life Sciences, Arlington
Heights, IL). Either densitometry or PhosphorImaging was used to
quantify the intensity of bands on autoradiograms.
MMC sensitivity assay.
The MMC growth inhibition assay was performed by exposing lymphoblast
cultures to a range of MMC concentrations in the presence or absence of
His-tagged recombinant proteins. Cell numbers were obtained by either
total cell counts using a Coulter (Hialeah, FL) counter or
Trypan blue exclusion and hemocytometer as described.14
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RESULTS |
Construction and in vivo function of chimeric protein.
IL-3 is a 133-amino acid glycoprotein that is produced mainly by
activated T cells. Its predicted tertiary structure consists of 4  -helices flanked by exposed ends. Sites on helix A and helix D
constitute the binding domain to high-affinity receptors.23 We constructed a fusion cDNA consisting of a histidine tag (His-tag) at
the amino-terminus, gibbon IL-3 (minus the signal peptide) at the
center, and full-length human FAC (minus the initiation codon) at the
carboxy terminus (called His-ILFAC; Fig 1).
We reasoned that placement of a fusion partner at the exposed carboxy
terminus of IL-3 would not result in steric hindrance. Despite these
considerations, it is possible that the resulting protein may be
intrinsically deficient, but the lack of an in vitro assay for FAC
function would not allow us to test this possibility before protein
replacement studies. We therefore asked whether or not the chimeric
cDNA can encode a functional polypeptide in vivo. We placed an
initiation codon with a Kozak consensus sequence at the 5 end of
the tripartite insert and subcloned it into the mammalian episomal
expression vector pDR2. Stable expression of this construct in HSC536
cells completely corrected the sensitivity of these cells to MMC.
Therefore, this construct is comparable in its in vivo activity to
wild-type FAC (Fig 2).
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| Fig 1.
Schematic structures of His-ILFAC and His-FAC. The
structures are drawn approximately to scale. The restriction sites and
sequence (BamHI site in italics) of the IL-3-FAC junction are
indicated. The last two codons for IL-3 at the carboxy terminus, a
two-residue linker, and the first two mature codons of FAC are also
shown. Hexahistidine tag (filled block), mature gibbon IL-3 (striped
block), FAC (open block).
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| Fig 2.
Activity of His-ILFAC transgene in vivo. HSC536
lymphoblasts transfected with pDR2-His-ILFAC or pDR2-FAC were selected
for stable expression of the episomal vectors by Hygromycin B, followed
by analysis of their growth in the presence of MMC. Growth inhibition
was measured by cell counting after 3 days (approximately 3 cell
divisions) of continuous exposure to MMC and compared with untreated
cells. The final cell count of untreated cultures was set at 100%. The
mean values of duplicate sets of experiments are shown.
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Expression and purification of His-ILFAC.
We also generated His-tagged FAC and cloned both His-FAC and His-ILFAC
into the prokaryotic expression vector pQE9. Expression of His-FAC and
His-ILFAC in E coli generated the expected 63-kD and 75-kD
fusion proteins, respectively (Fig 3A).
These fusion proteins were immobilized on nickel-agarose, washed, and
eluted. Additional purification of His-ILFAC included size exclusion
chromatography over Superdex-75 (Fig 3B). Western analysis confirmed
that the 75-kD protein is immunoreactive with affinity-purified
polyclonal antibodies directed against FAC. The final yield of
partially pure His-ILFAC protein was approximately 0.25 mg from 4 L of
induced bacterial culture.
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| Fig 3.
Expression and purification of His-FAC and His-ILFAC. (A)
Lysates from uninduced (lanes 1 and 4) or induced (lanes 2 and 5) E
coli or solubilized proteins (lanes 3 and 6) that were immobilized
on Ni2+-agarose, eluted with acid buffer, and analyzed by
10% SDS-PAGE and Coomassie blue staining. (B) Purification of
His-ILFAC by size exclusion chromatography. Soluble His-ILFAC obtained
after Ni2+-agarose chromatography was resolved further
over Superdex-75. Fraction 1, wash buffer; fractions 2 and 3, void
volume containing protein peaks as assessed by chromatography tracing
(data not shown); fraction 4, void volume containing no protein as
predicted by the flat chromatography tracing (data not shown). A 50-kD
protein copurifying with His-ILFAC, His-IL FAC, most likely results
from premature termination of translation as it is immuoreactive with
an anti-His antibody. However, carboxy-terminal degradation cannot be
excluded.
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Uptake and intracellular turnover of His-ILFAC by cells expressing
IL-3 receptors.
High-affinity binding sites for IL-3 are found on immature
CD34+ multipotential hematopoietic cells, myeloid cells, as
well as some B cells.23-25 We tested FA group C
lymphoblastoid cells (HSC536 and GM4510) as well as HSC72 and HSC230
cells of other FA subtypes for the expression of the IL-3 receptors and by reverse-transcription (RT)-PCR. Both cell lines tested
showed expression of the chain but not the chain (data not
shown). To determine whether these cells are capable of binding and
internalizing His-ILFAC, we incubated HSC536 lymphoblasts with 10 µg/mL His-ILFAC and His-FAC simultaneously for 10 minutes at 4°C,
washed in cold PBS, and shifted the temperature to 37°C to allow
internalization. After 5 minutes, cells were washed and lysed, and the
lysate was analyzed by immunoblotting with anti-FAC antibody.
Immunoreactive His-ILFAC of ~75-kD size was clearly detected under
these conditions (Fig 4). An apparently prematurely truncated His-ILFAC, His-IL FAC, also internalized. By
contrast, very little if any cell-associated His-FAC appeared to
internalize. After the initial 5-minute pulse, the half-life of
His-ILFAC was estimated by chase of the internalized protein for
various time intervals and found to be approximately 60 minutes, similar to that of transfected FAC (40 to 45 minutes).21
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| Fig 4.
Uptake and turnover of His-ILFAC. His ILFAC and His-FAC
(10 µg/mL of cells; lane 1) were bound to HSC536 lymphoblasts at
4°C, internalized by warming to 37°C, and the intracellular
fate of the fusion proteins was assessed by Western analysis with
anti-FAC antibody after 5 minutes (lane 3), 15 minutes (lane 4), 30 minutes (lane 5), 60 minutes (lane 6), 120 minutes (lane 7), and 240 minutes (lane 8) postbinding times. Lane 2, no recombinant protein
added.
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Receptor-mediated ligand endocytosis.
Internalization of His-ILFAC was inhibited when the incubated cells
were kept at 4°C (Fig 5).
Internalization was also inhibited by addition of recombinant human
IL-3 (10 µg/mL; gift of Dr GD Longmore, Washington University, St
Louis, MO) to the medium (Fig 6). The lack
of complete inhibition may relate to inefficient stripping of surface
IL-3 receptors with trypsin, as recognized previously.26 By
contrast, no inhibition of His-ILFAC uptake was seen when His-FAC or
murine IL-3 (WEHI conditioned medium) were used as competitors. Taken
together, these data strongly support the notion that His-ILFAC is
internalized by human lymphoblastoid cells by receptor-mediated
endocytosis. It is conceivable, however, that IL-3 receptors bound to
ligands could recycle back to the cell surface and release IL-3 into
the medium, which could create a futile cycle and result in a lower
effective intracellular concentration of His-ILFAC. To exclude this
possibility, 2 × 106 cells were incubated with
His-ILFAC for 10 minutes at 37°C, washed with PBS, and then allowed
to incubate in RPMI-10% FCS for up to 1 hour. The medium was then
clarified of cell debris and analyzed by immunoblotting. There was no
detectable His-ILFAC in the medium (data not shown). The lack of
extrusion into the medium suggests that, once inside the cell,
His-ILFAC remains confined to the intracellular compartment.
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| Fig 5.
Temperature-dependent uptake of His-ILFAC. HSC536
lymphoblasts were incubated with His-ILFAC for the indicated times at
either 37°C or 4°C were lysed and analyzed by immunoblotting
with anti-FAC antibody. Light units obtained by PhosphorImaging of the
immunoblot are given as percentages of the value at 10 minutes
(100%).
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| Fig 6.
Inhibition of His-ILFAC uptake. His-ILFAC internalization
by HSC536 lymphoblasts was inhibited by addition of recombinant human
IL-3 (10 µg/mL) to the medium, and inhibited further by pretreatment
of intact cells with 0.25% Trypsin for 10 minutes before incubation
with His-ILFAC (10 µg/mL). No inhibition was seen with His-FAC at 50 µg/mL (ie, 5-fold higher concentration than His-ILFAC) or with murine
IL-3 (WEHI conditioned medium at a concentration 10-fold higher than
that required for growth of the IL-3-dependent cell line HCD57). The
mean of three independent measurements and standard error of the mean
are shown.
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Correction of MMC sensitivity in FA group C cells.
The effect of His-ILFAC on cell survival was evaluated in HSC536 cells
exposed to MMC. Approximately 1 × 104 cells were
incubated with different concentrations of MMC for 5 days in the
presence or absence of His-ILFAC, and viable cells were quantified by
Trypan blue exclusion. Cells supplemented with His-ILFAC at a
concentration of 1 µg/mL, added at the beginning of the assay and
supplemented daily, were significantly more resistant to MMC than
control cells (Fig 7) and comparable to
HSC536 cells stably expressing wild-type FAC. His-ILFAC also protected
GM4510 cells from MMC cytotoxicity (data not shown). By contrast, under those conditions His-FAC at 1 µg/mL or recombinant human IL-3 at 1 µg/mL had no appreciable effect. Neither His-ILFAC nor His-FAC were
able to protect HSC72 and HSC230 lymphoblasts from the cytotoxicity of
MMC (data not shown). These data show that exogenous supplementation with His-ILFAC can protect FA group C cells from the toxicity of MMC to
an extent similar to that of transfected FAC.
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| Fig 7.
Complementation of FA group C cells with FAC
molecules. Trypan blue exclusion was used to assess cellular viability
of HSC536 lymphoblasts incubated in the presence of MMC continuously
for 5 days. HSC536-FAC, HSC536 lymphoblasts stably transfected with
wild-type FAC. Treatments with exogenous protein included His-ILFAC (1 µg/mL), His-FAC (1 µg/mL), and recombinant human IL-3 (1 µg/mL) added to the culture medium of HSC536 cells daily
for 5 days. Each experiment was performed in
triplicate. The mean and standard error of the mean are shown.
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DISCUSSION |
As an alternative to gene transfer, we have attempted to introduce the
FAC protein into hematopoietic cells by exploiting the interaction of
IL-3 with its receptor. In view of the difficulties associated with
conventional methods of gene transfer (eg, by transduction with viral
vectors) into hematopoietic cells, it seems reasonable to explore
alternative methods with the eventual goal of generating clinically
useful reagents.
Both murine and human IL-3 receptors are internalized upon binding
IL-3, followed by digestion of IL-3 within endosomes.26,27 The property of IL-3 receptors to undergo endocytosis upon ligand binding appears to be a general property as it is also observed in
other members of the cytokine receptor superfamily. Although the IL-3
ligand-receptor pair was used in this study primarily because of the
availability of mutant lymphoblasts that expressed the receptor, it may
be possible to select other ligand-receptor pairs and target additional
subsets of hematopoietic cells. Indeed, recent studies using analogous
strategies have included the targeting of cytokine receptor components
as fusion proteins with the constant region of IgG1 to Fc
receptors on CD34+ cells and surface-modified retroviruses
to cytokine receptors.28-31 The restricted expression of
IL-3 receptors on immature hematopoietic cells primarily, specificity
of binding to IL-3, and the ability of IL-3 receptors to internalize
bound ligands led us to use this ligand-receptor pair for the delivery
of FAC into hematopoietic cells. The His-ILFAC fusion protein was
expressed in E coli and purified to greater than 80%
homogeneity by two simple chromatographic techniques. Competition
experiments with exogenous IL-3 and stripping of IL-3 receptors from
the cell surface showed a significant inhibition in the uptake of the
His-ILFAC protein. As expected, human IL-3 but not murine IL-3 was able
to act as a competitor of His-ILFAC. Along with the observation that
this internalization is temperature-dependent, our results show that
His-ILFAC is internalized by receptor-mediated endocytosis.
We believe that several factors were responsible for the success of the
experiments described herein, which may not necessarily be extrapolated
to other systems. First, we reasoned that the inclusion of a tag at the
termini of the IL-3 and FAC proteins would lead to correct folding and
function of both domains of the fusion protein. The predicted tertiary
structure of IL-3 suggests that modifications of the amino or carboxy
termini may be tolerated well and would not interfere with the binding
domain established by the -helical structures.23 With
regard to FAC, we knew that attachment of a tag at the amino terminus
would not interfere with its function.12,14 By these
rudimentary considerations we predicted that a fusion protein
constructed in the His-ILFAC arrangement may fold correctly and
preserve the function of both IL-3 and FAC. Other attempts to generate
functional fusion proteins (eg, to target tumor cells with
antibody-toxin or antibody-reporter conjugates) have relied on the
placement of long linkers between fusion partners. Here, only two
additional amino acids were introduced between IL-3 and FAC, and even
those residues may be dispensible. Second, the availability of a simple
functional readout made it possible to test both the activity of the
transgene and the fusion protein in parallel. This was an important
attempt to distinguish partially functional from fully functional
fusion proteins. However, we cannot exclude the possibility that the
FAC domain of His-ILFAC folds correctly only after entry into cells.
Third, FAC is normally expressed at very low levels in most mammalian
cells.12 For functional complementation, a relatively small
amount of protein targeted to the appropriate cellular compartment may
be sufficient to give phenotypic correction. Conversely, the
demonstration that FAC overexpression causes no obvious toxicity in
transgenic mice indicates that the therapeutic-to-toxic ratio of FAC is
highly favorable.32 Fourth, the hydrophobic nature of FAC
may be an important determinant which facilitates its exit from
endosomes after endocytosis. In this way, a prolonged exposure to the
acidic milieu of endosomes and excessive proteolysis may be
circumvented. Finally, the size of a fusion protein may be a
significant impediment to its expression in various host cells and
penetration into cells.33 The relatively small size of the
His-ILFAC fusion protein allows for efficient expression in bacteria
and potentially good access to hematopoietic progenitor cells.
There are also a number of potential shortcomings with the projected
use of this or similar reagents for protein replacement therapy. Chief
among them is the paucity of cytokine receptors on hematopoietic
progenitor cells. A higher receptor density may be more favorable. We
have designed our expression cassette to enable the rapid exchange of
IL-3 with other ligands. The short half-life of FAC also may be a
limiting factor. Although it is reassuring that the intracellular
half-life of His-ILFAC is at least comparable to that of transfected
wild-type FAC cDNA,20 longer-acting forms of FAC may
function in a more efficient manner. Finally, we do not know whether or
not FAC is required during particular stages of the cell cycle. If FAC
acts predominantly at a particular stage, pulse delivery of the fusion
protein may be sufficient to achieve complementation. Alternatively,
there may be a constant requirement for FAC if it is needed throughout the cell cycle. Hence, the precise time of FAC supplementation in the
context of cell turnover is unclear. In our cytotoxicity assay (Fig 7),
complementation was achieved by daily addition of His-ILFAC to cell
cultures. In preliminary experiments, His-ILFAC at 100 µg/mL (10-fold
higher concentration than that used in the previous regimen) added once
at the beginning of the 5-day exposure period to MMC was also able to
confer resistance to MMC (data not shown). Clearly, the optimum
delivery schedule needs to be established. In future experiments we
hope to define the temporal need for FAC activity, compare other
receptor-ligand pairs, and identify FAC residues that, through in vitro
mutagenesis, may lead to longer active forms of this molecule.
To our knowledge, this is the first report of an attempted restoration
of function in a mendelian disorder by a protein engineered to undergo
receptor-mediated endocytosis. It is functionally equivalent to enzyme
replacement therapy for several inborn errors of metabolism using
mannose-6-phosphate-dependent uptake of proteins into
cells.34 In the absence of natural receptors for many
intracellular proteins, the engraftment of IL-3 or possibly other
cytokines may facilitate the delivery of therapeutic proteins into
hematopoietic cells.
| |
ACKNOWLEDGMENT |
We thank Drs Steve Clark (Genetics Institute, Cambridge, MA) and Greg
Longmore (Washington Universdity, St Louis, MO) for reagents. The early
part of this work was performed at the Brigham and Women's Hospital.
The physical and intellectual support provided by that institution is
gratefully acknowledged.
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FOOTNOTES |
Supported by a Translational Research Award from the Leukemia Society
of America.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hagop Youssoufian, MD, Department of
Molecular and Human Genetics, Baylor College of Medicine, One Baylor
Plaza, S840, Houston, TX 77030; e-mail: hagopy{at}bcm.tmc.edu.
| |
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Abstract
Introduction