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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3193-3198
Reversal of Metabolic Block in Glycolysis by Enzyme Replacement in
Triosephosphate Isomerase-Deficient Cells
By
Art Ationu,
Ann Humphries,
Michel R.A. Lalloz,
Roopen Arya,
Barbara Wild,
Joanne Warrilow,
Jennifer Morgan,
Alastair J. Bellingham, and
D. Mark Layton
From the Department of Haematological Medicine, Guy's, King's, and
St Thomas' School of Medicine; and the Muscle Cell Biology Group,
Medical Research Council, Imperial College School of Medicine,
Hammersmith Hospital, London, UK.
 |
ABSTRACT |
Inherited deficiency of the housekeeping enzyme triosephosphate
isomerase (TPI) is the most severe clinical disorder of glycolysis. Homozygotes manifest congenital hemolytic anemia and progressive neuromuscular impairment, which in most cases pursues an inexorable course with fatal outcome in early childhood. No effective therapy is
available. Hitherto specific enzyme replacement has not been attempted
in disorders of glycolysis. Primary skeletal muscle myoblasts and
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines
generated from homozygous TPI-deficient patients were cultured in the
presence of exogenous enzyme or cocultured with human K562
erythroleukemia cells as an exogenous source of TPI. Uptake of active
enzyme by TPI-deficient cells resulted in reversal of intracellular
substrate accumulation, with a reduction in dihydroxyacetone phosphate
(DHAP) concentration to levels seen in TPI-competent cells. Evidence of
successful metabolic correction of TPI deficiency in vitro establishes
the feasibility of enzyme replacement therapy, and has important
implications for the potential role of allogeneic bone marrow
transplantation and gene therapy as a means of sustained delivery of
functional enzyme in vivo.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
TRIOSEPHOSPHATE ISOMERASE (TPI, EC
5.3.1.1) catalyzes the interconversion of dihydroxyacetone phosphate
(DHAP) and glyceraldehyde-3-phosphate in the Embden-Meyerhof pathway,
with the reaction favoring formation of DHAP by a ratio of
20:1.1 TPI deficiency is an autosomal recessive multisystem
disorder characterized by congenital hemolytic anemia, progressive
neuromuscular dysfunction, susceptibility to bacterial infection, and
cardiomyopathy. The majority of affected children fail to survive
beyond 5 years of age.1-3 Homozygotes exhibit markedly
reduced enzyme activity in all tissues studied, accompanied by
metabolic block in glycolysis with intracellular accumulation of DHAP,
particularly in red blood cells, which lack the capacity to metabolize
DHAP in the glycerophosphate shuttle via  -glycerophosphate
dehydrogenase.1,2 Genetic studies of multiple unrelated
families have shown that a single mutation, G to C transversion at
codon 104, accounts for 80% of mutant alleles, reflecting a founder
effect.3 The substitution of aspartate for glutamate at
residue 104, which lies at the subunit interface of the TPI dimer,
results in loss of activity due to instability of the mutant enzyme.
Several other causative mutations (8 missense, 2 nonsense, and 1 frameshift) distributed throughout the TPI gene have been
defined.4-7 Progress towards understanding the molecular
basis of TPI deficiency has overcome the limitations of biochemical
assessment for in utero diagnosis.8,9 However, there is no
effective therapy for the generalized manifestations of the disease.
Metabolic correction of several inborn errors, including severe
combined immunodeficiency due to adenosine deaminase deficiency, X-linked adenoleukodystrophy, and the lysosomal storage disorders Gaucher disease, mucopolysaccharidosis (types I, II, and VII), and
metachromatic leukodystrophy, has been achieved by enzyme replacement
therapy and bone marrow transplantation.10-15 To date, neither approach has been attempted in TPI deficiency.
Heterozygotes have approximately 50% normal TPI activity, but manifest
no evidence of metabolic block or clinical effects, implying this level
of enzyme activity is sufficient to maintain normal metabolic
function.5-8 Restoration of enzyme activity to comparative
levels is therefore expected to produce substantial clinical benefit in
homozygous TPI deficiency. The capacity of enzyme-replete cells to
secrete functional enzyme that can be captured by deficient cells in
cross-correction studies provides a basis for investigating the
feasibility of intracellular enzyme delivery. Primary skeletal muscle
myoblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell
lines derived from patients with homozygous TPI deficiency manifest the
biochemical characteristics of TPI deficiency1,2,16,17 and
thus provide a model system for the disease.
In a preliminary study, transfer of functional enzyme in vitro from
cells with normal TPI activity to lymphoblastoid cells derived from a
TPI-deficient patient was demonstrated.16 In the present
study, the feasibility of somatic correction was studied in primary
skeletal muscle myoblasts and lymphoblastoid cells derived from
TPI-deficient patients with different mutant genotypes. Restoration of
intracellular enzyme activity with reduction in DHAP to normal levels
was achieved in deficient primary skeletal muscle myoblasts and
lymphoblastoid cells cultured in the presence of exogenous human plasma
or purified rabbit muscle TPI, and after coculture with human K562
erythroleukemia cells as a renewable source of functional enzyme.
Reversal of the metabolic defect in deficient cells, in particular
primary skeletal muscle myoblasts, confirms the feasibility of enzyme
replacement therapy for TPI deficiency and has implications for the
potential role of allogeneic bone marrow transplantation and gene
therapy as strategies for sustained enzyme delivery in vivo.
| |
MATERIALS AND METHODS |
Patients and samples.
The study was approved by the institutional ethics committee. Muscle
biopsy tissue and blood samples of affected children were obtained with
informed parental consent. Details of the 3 TPI-deficient patients
studied are listed in Table 1. Patient A is
homozygous for the codon 104 mutation (Glu104Asp). Patients B and C are
compound heterozygous for codon 104 and 41 (Cys41Tyr) mutations and
codon 104 and 170 (Ile170Val) mutations, respectively. All patients
presented with congenital hemolytic anemia and exhibit marked reduction
in erythrocyte enzyme activity and DHAP accumulation.
Cell lines.
EBV-transformed lymphoblastoid cell lines derived from patients A, B,
and C, a heterozygote (D; mother of patient A), and a normal subject
were maintained in culture at 0.5 to 1 × 106 cells/mL
in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mmol/L
glutamine, 50 µg/mL streptomycin, and 1 mg/mL penicillin (GIBCO Life
Technologies, Glasgow, UK). Human K562 erythroleukemia cells were
maintained in the same medium. Human muscle cell line HS94MU (European
Collection of Animal Cell Cultures, Wiltshire, UK) was maintained in
culture at 1 × 105 cells/mL in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 20% FCS, 2 mmol/L glutamine,
50 µg/mL streptomycin, and 1 mg/mL penicillin. Human astrocytoma cell
line MOG-G-UVW (European Collection of Animal Cell Cultures [ECACC])
was maintained in culture at 2 × 104 cells/mL in
Ham's F10 nutrient medium mixed with DMEM (1:1) supplemented with 10%
FCS, 2 mmol/L glutamine, 50 µg/mL streptomycin, and 1 mg/mL
penicillin. All cell lines were cultured at 37°C, 5%
CO2, 95% air and used for experiments after the third passage.
Primary skeletal muscle myoblasts.
Quadriceps muscle tissue from patient A was rapidly transferred after
open biopsy to the tissue culture laboratory where primary skeletal
muscle myoblasts were isolated. Muscle tissue was minced finely in
phosphate-buffered saline (PBS) containing 25 µg/mL fungizone
(amphotercin B; Flow Laboratories, Lichfield, Staffordshire, UK) and
1% streptomycin/penicillin to obtain fragments of approximately 1 to 2 mm3 in size. These were washed 3 times in growth medium
(GM: Ham's F-10 nutrient medium supplemented with 20% FCS, 2%
glutamine, and 1% streptomycin/penicillin), after which they were
resuspended in 0.5 mL GM. Approximately 5 to 10 explants were
transferred with GM to a tissue culture flask coated with sterile
0.01% gelatin and incubated at 37°C, 5% CO2, 95%
air. As soon as cells migrated from explants, the cultures were
trypsinized (0.25% trypsin-EDTA) at 37°C and cells and explants
were replated separately. Explanted muscle cells were
maintained in culture at 1 × 105 cells/mL in GM and
cultured in a humidified environment (37°C, 5% CO2,
95% air). Preplating of the primary cultures was performed once to
separate fibroblasts and myoblasts. Confluent myoblasts were
subcultured and used for all experiments after the third passage. The
myogenicity of explanted primary skeletal muscle cells was determined
by the capacity for myotube formation in vitro (fusion index) and
expression of the muscle-specific protein, desmin. The fusion index was
82.9%, and 91.2% of cells were desmin-positive.
Culture of TPI-deficient cells in the presence of plasma or purified
enzyme.
Fresh-frozen plasma (FFP) was initially used as a source of normal
human TPI. TPI activity was determined in FFP (range, 16 to 79.8 U/mL;
n = 4) obtained from the hospital blood bank. Serum-free medium (GIBCO
Life Technologies, Europe) was mixed with FFP to obtain a TPI
concentration of 40 U/mL. Lymphoblastoid cells (1 × 105 cells) from patient A were cultured for 21 hours at
37°C, 5% CO2 , 95% air in the FFP-treated medium.
Control lymphoblastoid cells were cultured under similar conditions in
untreated serum-free medium. To exclude the possibility that any
increase of intracellular TPI activity in lymphoblastoid cells cultured
in FFP is dependent on protein synthesis, parallel experiments were
performed in which cycloheximide (10 µg/mL) was added to the culture
medium18 and incubated for 21 hours at 37°C, 5%
CO2, 95% air.
Lysates were prepared by freezing cells in liquid nitrogen and thawing
on ice. After culture, lymphoblastoid cells were washed 3 times in
ice-cold PBS and lysed by 3 cycles of freezing and thawing. Lysates
were centrifuged (3,000 rpm, 10 minutes, 4°C) and the supernatant
collected on ice. An aliquot of the supernatant was assayed for TPI
activity and total cellular protein. For determination of DHAP levels,
aliquots of the supernatant were deproteinized immediately by mixing
with ice-cold 5% perchloric acid, incubated on ice for 20 minutes, and
centrifuged (3,000 rpm, 10 minutes, 4°C). The supernatant was
neutralized with 3 mol/L K2CO3
solution containing 1 mol/L Tris. After centrifugation, the clear
supernatant was stored at  70°C and assayed within 24 hours.
Primary skeletal muscle myoblasts (1 × 105 cells)
from patient A, lymphoblastoid cells (1 × 105 cells)
from patients A, B, C, a heterozygote, D, a nondeficient subject, and
cell lines HS94MU (1 × 104 cells) and MOG-G-UVW (1 × 104 cells) were incubated for 24 hours in 1 mL
serum-free medium containing 50 U of purified rabbit muscle TPI
(Boehringer Mannheim, Mannheim, Germany) sterilized by filtration
(Millipore; Sartorius Ltd, Epsom, Surrey, UK). Control cells were
cultured under identical conditions in serum-free medium alone.
At the end of the culture period, primary skeletal muscle myoblasts,
human muscle, and astrocytoma cell lines were washed 3 times in
ice-cold PBS, trypsinized (0.25% trypsin-EDTA) at 37°C for 10 minutes, frozen in liquid nitrogen, and homogenized on ice for 2 minutes. Homogenates were centrifuged and an aliquot of supernatant was
assayed for TPI activity and total cellular protein. Aliquots of
supernatant for DHAP determination were immediately deproteinized as
described earlier.
Coculture of TPI-deficient cells and K562 erythroleukemia cells.
Uptake of exogenous TPI by deficient primary skeletal muscle myoblasts
from patient A and lymphoblastoid cells from patients A, B, and C was
examined in a coculture model using human K562 erythroleukemia cells as
a source of functional enzyme.16 K562 cells were plated
into 6-well cell-companion culture dishes (5 × 105
cells/well) containing serum-free medium. To prevent direct
cell-to-cell contact, primary skeletal muscle cells (1 × 105 cells/well) and lymphoblastoid cells (5 × 105 cells/well) were plated separately into inserts
containing a semipermeable membrane of 1 µm pore size
(Becton-Dickinson Labware, Oxford, UK). The cocultures were incubated
at 37°C, 5% CO2, 95% air for 24 hours. Control muscle
and lymphoblastoid cells cultured in serum-free medium in the absence
of K562 cells were maintained under similar conditions.
DHAP levels in protein-free extracts.
To exclude the possibility that membrane adherent or encapsulated
enzyme contributed to the reduction in DHAP concentration observed in
cell lysates, lymphoblastoid cells (1 × 105 cells)
from patient A (Table 1) were cultured in serum-free medium (control)
or in the presence of exogenous rabbit muscle TPI (50 U/mL) and sampled
at 0, 3, 6, 9, 12, and 24 hours. Cultured cells were immediately
deproteinized by the addition of ice-cold 5% perchloric acid. The
deproteinized cell suspension was incubated on ice for 20 minutes,
centrifuged (3,000 rpm, 10 minutes, 4°C), and the supernatant
neutralized as described earlier and assayed for DHAP.
In parallel experiments, lymphoblastoid cells (1 × 105 cells) from patient A (Table 1) cultured in serum-free
medium (control) or in the presence of rabbit muscle TPI (50 U/mL) were
sampled at various intervals. Cultured cells were either deproteinized with 5% perchloric acid as described or lysed by immediately freezing cells in liquid nitrogen and thawing on ice. Aliquots of lysates were
deproteinized with 5% perchloric acid and neutralized. DHAP assays
were performed as describe earlier.
Assay of TPI activity and DHAP level.
TPI activity in FFP, culture medium, cell lysates, and homogenates was
determined according to the method recommended by the International
Committee for Standardisation in Haematology (ICSH) for quantification
of enzyme activity in erythrocytes.19,20 Briefly, culture fluid or cell lysates were added to a cuvette in the
presence of DL-glyceraldehyde-3-phosphate as substrate in an
-glycerophosphate dehydrogenase/NADH-coupled assay. Decrease in
optical density was measured at 340 nm, 30°C, 10 minutes. DHAP was
estimated by fluorimetric analysis as previously
described.8,16 Protein concentration in homogenates and
lysates was determined by the method of Bradford, with bovine serum
albumin as standard.21
Statistical analysis.
All results are given as the mean ± SD of 6 separate experiments in
triplicate, unless otherwise indicated. Student's t-test was
used to establish whether between-group differences were statistically significant.
| |
RESULTS |
Culture of TPI-deficient cells with plasma.
Initial experiments assessed the feasibility of enzyme replacement in
vitro with FFP. Lymphoblastoid cells from patient A (Table 1) cultured
in the presence of FFP as an exogenous source of enzyme showed a 5-fold
reduction in intracellular DHAP level (84 ± 35 µmol/L/µg
protein) with a concomitant rise in TPI activity from 37 ± 15 to
361 ± 33 U/µg protein (Fig 1A and B).
The corresponding DHAP level and TPI activity in lymphoblastoid cells
cultured in the absence of FFP were 388 ± 60 µmol/L/µg protein
and 30 ± 15 U/µg protein, respectively. At the end of the culture
period, TPI activity in the culture medium had decreased by
approximately 2-fold, whereas no TPI activity was detectable in the
control medium (Fig 1C). Lymphoblastoid cells (1 × 106 cells) cultured in the presence of FFP for 21 hours,
washed in PBS, and reintroduced into FFP-medium or serum-free medium
(control) for a further 24 hours, showed that the intracellular DHAP
level was 178 ± 48 µmol/L/µg protein in cells maintained in FFP
and 1,023 ± 380 µmol/L/µg protein in controls. The
corresponding TPI activity was 188 ± 10 U/µg protein and 21 ± 4 U/µg protein in treated and control cells, respectively.
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| Fig 1.
Reversal of metabolic defect in TPI-deficient
lymphoblastoid cells from patient A (Table 1). (a) Reduction in
accumulated intracellular DHAP. (b) Intracellular TPI activity. (c) TPI
activity in culture medium. Cells were cultured in serum free medium
with ( ) or without ( ) normal human plasma as described in
Materials and Methods.
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Cycloheximide-treated lymphoblastoid cells similarly showed increased
intracellular TPI activity (255 ± 45 U/µg protein) and a reduced
DHAP level (531 ± 105 µmol/L/µg protein) in the presence of FFP
when compared with untreated cells (TPI activity, 25 ± 10 U/µg
protein; DHAP, 1,606 ± 255 µmol/L/µg protein). This indicates that reversal of the metabolic block in deficient cells is not dependent on de novo protein synthesis.
Culture of TPI-deficient cells with purified TPI.
The ability of purified enzyme to reverse the metabolic defect in
TPI-deficient lymphoblastoid cells and primary skeletal muscle
myoblasts cultured in the presence of purified rabbit muscle TPI was
evaluated by estimating intracellular accumulation of DHAP and TPI
activity. After incubation with exogenous TPI, the intracellular DHAP
level and TPI activity observed in cell lines from patients A, B, and C
were comparable to those in cells derived from normal and heterozygous
subjects (Fig 2). The alterations in
intracellular TPI activity and DHAP level for lymphoblastoid cells
cultured in the presence of rabbit muscle TPI were highly significant
when compared with controls cultured in serum-free medium alone (Fig
2). When TPI-deficient primary skeletal muscle myoblasts were cultured
in the presence of purified rabbit muscle TPI, a 4- to 5-fold increase
in intracellular TPI activity (340 ± 65 U/µg protein),
accompanied by a reciprocal decrease in DHAP level (105 ± 30 µmol/L/µg protein), was observed. These figures are highly
significant (P < .001) when compared with control values (TPI
activity, 75 ± 25 U/µg protein; DHAP, 1,600 ± 315 µmol/L/µg protein), and equivalent to those in normal human primary
skeletal muscle myoblasts (TPI activity, 550 ± 60 U/µg protein;
DHAP, 80 ± 25 µmol/L/µg protein; n = 2).
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| Fig 2.
Reversal of metabolic block in TPI deficiency by
exogenous rabbit muscle TPI (RTPI). Lymphoblastoid cells derived from
TPI-deficient patients A, B, and C, a heterozygote, D, and a normal
subject were cultured in serum-free medium with or without exogenous
RTPI (50 U/mL) for 24 hours. Changes in intracellular enzyme activity
and DHAP level were measured as described in Materials and Methods.
Statistical significance: *P < .05, **P < .01, ***P < .001; NS, not significant.
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Uptake of TPI by other human cell lines.
Table 2 shows that human skeletal muscle
and astrocytoma cell lines cultured in the presence of exogenous rabbit
muscle TPI have an increased TPI activity when compared with their
respective basal level.
Coculture of TPI-deficient cells and K562 cells.
To investigate whether sustained correction of the metabolic defect in
TPI-deficient cells in vitro may be feasible, deficient lymphoblastoid
cells or primary skeletal muscle myoblasts were cocultured with K562
cells. Figure 3 shows results obtained when TPI-deficient lymphoblastoid cells from patients A, B, and C (Table 1)
were cocultured with K562 cells. After 24 hours in coculture, there was
a significant reduction in DHAP and a significant increase in
intracellular TPI activity when compared with lymphoblastoid cells
cultured in the absence of K562 cells (Fig 3). Cocultured deficient
primary skeletal muscle myoblasts showed a significant increase (P
< .001) in intracellular TPI activity (328 ± 75 U/µg protein) with a significant reduction (P < .001) in
DHAP level (93 ± 45 µmol/L/µg protein) when compared with
control values (TPI activity, 89 ± 30 U/µg protein; DHAP, 1,491 ± 250 µmol/L/µg protein).
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| Fig 3.
Reversal of metabolic block in TPI deficiency using a
coculture model. Lymphoblastoid cells derived from patients A, B, and C
were cocultured for 24 hours in the presence or absence (control) of
human K562 erythroleukemia cells in serum free-medium as described in
Materials and Methods. Statistical significance: *P < .05, **P < .01, ***P < .001.
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DHAP concentration in deproteinized extracts.
Fig 4 shows results obtained when
lymphoblastoid cells from patient A (Table 1) cultured in the presence
of rabbit muscle TPI were immediately deproteinized in perchloric acid.
There was a marked reduction in DHAP concentration for cells cultured
in the presence of exogenous TPI when compared to controls cultured in
serum free medium alone. These results demonstrate conclusively that
metabolic block is reversed through uptake of active enzyme by
TPI-deficient cells.
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| Fig 4.
Change in DHAP level for TPI-deficient lymphoblastoid
cells immediately deproteinized in 5% perchloric acid. Cells were
cultured in serum-free medium with () or without () exogenous
rabbit muscle TPI (50 U/mL) and processed as described in Materials and
Methods. Each point represents the mean of 1 experiment performed in
triplicate.
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To further eliminate the possibility that membrane adherent or
entrapped enzyme might contribute to the reduction in intracellular DHAP level in deficient cells cultured in the presence of TPI, DHAP
concentration in deproteinized cell extracts and cell lysates was
compared. No significant difference between the DHAP level in cell
lysates and that from deproteinized cell extracts was observed (Fig
5).
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| Fig 5.
Comparison between the change in DHAP level for
TPI-deficient lymphoblastoid cells immediately deproteinized in
perchloric acid () and cell lysates ( ). Cells were cultured in
the presence of exogenous rabbit muscle TPI (50 U/mL) and protein-free
extracts processed as described in Materials and Methods. Each point
represents the mean of 1 experiment performed in triplicate.
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| |
DISCUSSION |
Specific enzyme replacement therapy has hitherto not been attempted in
TPI deficiency or other inborn errors of glycolytic metabolism. TPI
deficiency is the most clinically severe defect of glycolysis and
typically results in death before the age of 6 years. In patients with
severe clinical phenotypes, no form of therapy has been shown to modify
the natural history of the disorder. Efforts to bypass the enzymatic
defect by methylene blue stimulation of the hexose monophosphate shunt
have proved unsuccessful and splenectomy did not alter the course of
the disease in one patient.2 The prospect for somatic
correction of TPI deficiency by enzyme replacement therapy was
therefore studied. Alterations in intracellular DHAP concentration and
TPI activity were monitored to evaluate the capacity of exogenous
enzyme to reverse metabolic block in TPI-deficient cells. The results
clearly demonstrate increased intracellular TPI activity with a marked reduction in DHAP accumulation in deficient cells treated with exogenous TPI or cocultured with donor cells as a renewable source of
functional enzyme.
While TPI localizes in the cytosol, enzyme activity is detectable in
plasma.7,8,17 The origin of extracellular TPI is uncertain,
but is likely to reflect diffusion or secretion from blood cells. This
prompted examination of the potential role of human plasma as a vehicle
for delivery of exogenous enzyme to TPI-deficient lymphoblastoid cells
and whether correction of TPI deficiency in vitro could be achieved at
a concentration within the physiologic range found in FFP.
TPI-deficient lymphoblastoid cells cultured in the presence of FFP
showed a nadir in intracellular DHAP level coincidental with maximal
TPI activity. Partial reversal of this effect with time was seen due to
gradual depletion of enzyme in medium (Fig 1). This suggests frequent
or continuous delivery of enzyme may be necessary to achieve
therapeutic benefit in vivo.22 Functional restoration of
glycolysis in vitro was confirmed in TPI-deficient lymphoblastoid cells
cultured in the presence of rabbit muscle TPI (Fig 2). DHAP was reduced
to a level comparable to that seen in nondeficient cells.
Notably, reversal of metabolic block in lymphoblastoid cells was
observed for each of the 3 TPI-deficient patients studied, irrespective
of the causative mutation. Reaccumulation of DHAP and reversion of TPI
activity to basal levels were observed when medium to which exogenous
enzyme had been added was replaced with medium lacking the enzyme.
Patients homozygous for TPI deficiency have less than 20% residual
enzyme activity in muscle cells.23 The consequence of impaired glycolysis and defective mitochondrial metabolism has been
directly linked to the severe myopathy seen in homozygous TPI
deficiency.23 This study provides evidence for correction of the metabolic defect in TPI-deficient primary skeletal muscle myoblasts by exogenous enzyme. Furthermore, other cell types, including
nondeficient human muscle and astrocytoma cell lines (Table 2), as well
as lymphoblastoid cell lines from normal and heterozygous subjects (Fig
2), exhibited uptake of TPI from culture medium containing enzyme.
These observations imply existence of a transport mechanism that
permits the transfer of functional enzyme across the cell membrane. The
basis for this remains to be elucidated. Taken in conjunction, these
results establish the feasibility of intracellular enzyme replacement
therapy in TPI deficiency.
The mechanism underlying the neuropathic effects of TPI deficiency,
which may include central and peripheral components, is unknown.
Disturbance of lipid metabolism secondary to accumulation of DHAP, a
precursor of ether lipids which are essential components of myelin, has
been postulated.24,25 Despite evidence that the metabolic
defect in TPI-deficient myoblasts and lymphoblastoid cells is
reversible, it remains to be determined whether enzyme replacement
would correct the prominent neurological manifestations of TPI
deficiency. Inability of functional enzyme to traverse the blood-brain
barrier and its relatively short half-life are likely to present
significant obstacles in vivo.
Provision of a deficient enzyme to neural cells by bone marrow derived
microglial cells after hemopoietic stem-cell transplantation ameliorates or arrests neurologic progression in some lysosomal and
peroxisomal disorders.10-15 Evidence of correction from in vitro models of secretion and recapture of functional enzyme generally correlates with a favorable outcome after bone marrow transplantation. Coculture of TPI-deficient primary myoblasts and lymphoblastoid cells
with human K562 erythroleukemia cells led to functional restoration of
glycolysis. Recent studies in which myeloid colonies (colony-forming
unit granulocyte-macrophage [CFU-GM]) from a TPI-deficient patient
were cultured in methyl cellulose-containing feeder leukocytes derived
from a normal human leukocyte antigen (HLA)-matched related donor
confirm hematopoietic cells are a viable source of functional enzyme
(Ationu A, Humphries A, Gordon M, et al, unpublished observations, December 1997). Collectively, these results have important implications for the possible role of allogeneic bone marrow transplantation as a
means of sustained delivery of TPI to neural and other somatic cells,
and provide a rationale for development of therapeutic approaches for
correction of the defect in TPI deficiency and potentially other
metabolic disorders of glycolysis associated with a severe clinical phenotype.
| |
ACKNOWLEDGMENT |
We are grateful to David McGonigle for technical assistance with
biochemical studies.
| |
FOOTNOTES |
Submitted February 12, 1999; accepted July 1, 1999.
Supported by grants from the James Stewardson TPI Research Trust and
Medical Research Council.
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 Art Ationu, MSc, PhD, Department of
Haematological Medicine, Guy's, King's, and St Thomas' School of
Medicine, Bessemer Rd, London, SE5 9PJ, UK.
| |
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