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GENE THERAPY
From the St Jude Children's Research Hospital,
Memphis, TN.
Protective protein/cathepsin A (PPCA), a lysosomal
carboxypeptidase, is deficient in the neurodegenerative lysosomal
disorder galactosialidosis (GS). PPCA Defective genes encoding specific lysosomal
hydrolases are responsible for more than 40 disorders of the
metabolism, known as lysosomal storage diseases (LSDs).1
One of the glycoproteinoses, the autosomal recessive galactosialidosis
(GS),2,3 results from mutations in the PPCA
gene,4 causing a secondary deficiency of Enzyme replacement that ameliorates or reverses systemic and neurologic
defects is the goal of curative treatment for LSDs. This strategy is
based on the observation that soluble enzyme precursors secreted by one
cell type can be internalized via receptor-mediated endocytosis by
deficient cells with consequent resolution of toxic catabolite
accumulation, that is, correction "in trans." BM progenitor cells
are an attractive source of corrective enzyme because of their
potential to repopulate the recipient and to supply functional enzyme
to cells in affected organs, including the central nervous system
(CNS).6 Allogeneic bone marrow transplantation (BMT) and
syngeneic BMTs in affected patients and animal models effectively ameliorate visceral and bony lesions7-13; however, diseases
with early, predominantly CNS involvement respond
poorly.8,14 Allogeneic BMT is still limited by
difficulties in finding suitable HLA-compatible donors, high
rates of nonengraftment, severe graft-versus-host disease, and other
causes of transplantation-related morbidity and mortality.
The use of autologous hematopoietic progenitor cells (HPCs) that are
genetically engineered to express a therapeutic gene could, in
principle, circumvent some transplantation-associated obstacles. We
have recently proven the feasibility of a BMT approach in our murine GS
model. Early in life, PPCA We have now tested whether genetically modified PPCA Cell lines and vector construction
Generation of an ecotropic virus producer line
BMT
Determination of GFP expression in PBCs Recipients were bled by orbital sinus puncture at 1, 3, 6, and 8 to 10 months after BMT. Blood (20 µL) was collected in 1 mL cold phosphate-buffered saline (PBS) for FACS analysis of erythrocytes and platelets. For analysis of lymphocytes, erythrocytes were lysed in Gay solution and propidium iodide was added.23Enzyme activity assay Tissues were homogenized in water. Cathepsin A activity was measured with the synthetic dipeptide substrate Z-Phe-Ala as described earlier.24 Total protein concentration was determined with the bicinchoninic acid reagent (Pierce Chemical, Rockford, IL).Histochemical analysis Antibodies against the 32-kd subunit of human PPCA ( -32) were
raised in rabbit and affinity purified against the human
protein.25 This antibody was shown to selectively
recognize the human PPCA protein and does not cross-react with the
endogenous murine PPCA.16 Paraffin-embedded tissue
sections were deparaffinized and hydrated; antigen retrieval was
accomplished by boiling the sections in 0.1 M citrate, pH 6.0. After a
20-minute blocking process, the sections were incubated
overnight with either -32 or anti-GFP (-GFP, Clontech
Laboratories, Palo Alto, CA) antibodies followed by washing and
incubation with goat-antirabbit IgG secondary antibodies (Pharmingen,
San Diego, CA) for 2 hours at room temperature. Antigen-antibody complexes were detected with the ABC horseradish peroxidase system, which uses a VIP (purple) or diaminobenzidine (brown) substrate (Vector, Burlingame, CA). For PEP19 staining, serially sectioned, cerebella were processed and incubated with anti-PEP19
antibodies26 (a kind gift of Dr James Morgan, Developmental
Neurobiology, St Jude Children's Research Hospital) as above.
Purkinje cell count Counting of Purkinje cells was performed following the method described in Smeyne and Goldowitz.27
Expression of retrovirally encoded PPCA in GS BM cells To investigate whether genetically modified HPCs can correct the murine GS phenotype, we constructed a MSCV-based bicistronic vector containing PPCA cDNA that was linked by an internal ribosomal entry site to the gene encoding the GFP marker (MSCV-PPCA, Figure 1A). An identical vector carrying only the GFP gene was used as a control (MSCV-GFP). We first determined that the total number of BM cells harvested from PPCA /
donors, aged 2 to 6 months, was similar to that of cells from wild-type, age-matched mice and that the different lineages were correctly represented. Total PPCA/ BM was
then transduced with either MSCV-PPCA or MSCV-GFP ex vivo, to assess the transducibility of deficient cells versus normal BM.
In 2 pilot experiments performed before BMT, the transduction efficiency of PPCA/ BM cells, calculated on
the basis of GFP expression, was 15% and 20% with the MSCV-PPCA
vector, and 19% and 39% with the MSCV-GFP vector. In parallel, a
15-fold increase in cathepsin A activity was measured in transduced
PPCA/ BM cells compared to untreated cells or
cells transduced with the MSCV-GFP vector. We also assessed the level
of cathepsin A activity in lysates of clonogenic progenitor colonies
that were positive for GFP as visualized by fluorescence microscopy.
Cathepsin A activity was more than 100-fold higher in
MSCV-PPCA+ colonies than in MSCV-GFP-transduced colonies.
Correction of the murine GS phenotype by genetically modified
PPCA / BM cells transduced with
either MSCV-PPCA or MSCV-GFP into lethally irradiated, 3- to 6-week-old
GS mice. In 5 independent transplantation experiments, the transduction
efficiency of either the MSCV-PPCA or the MSCV-GFP retrovirus was
calculated on the basis of GFP expression in FACS-sorted cells,
immediately before transplantation. With the exception of the second BM
transduction experiment (see "Materials and methods"), the
transduction efficiency of the MSCV-PPCA virus was 28% (experiment
[exp] 1); 23% (exp 3); 19% (exp 4); and 17% (exp 5). The
transduction efficiency of the MSCV-GFP virus ranged from 19% to 44%.
GFP-expressing cells of the erythroid, myeloid, or lymphoid lineage
were detected by FACS analysis of peripheral blood samples, collected
at different time points after transplantation. Regardless of the
vector used, the percentage of gated cells expressing GFP varied
between 18% and 40% in erythrocytes, 20% and 61% in platelets, and
20% and 56% in lymphocytes (Figure 1B). To estimate the levels of the
therapeutic enzyme in different transplanted mice, cathepsin A activity
was assayed in tissue homogenates from organs of recipients at various
time points after BMT. For as long as 10 months after treatment,
increased cathepsin A activity was detected in most tissues; the
highest levels were measured in spleen, BM, and thymus, but also liver,
kidney, and heart had persistent and increased activity compared to the
knockout or BMT-GFP-treated mice (Figure 1C). Cathepsin A activity in
total brain lysates, which is usually low also in wild-type samples, was only marginally increased and varied among animals receiving transplants, probably because of the uneven distribution of engrafted cells that expressed the corrective enzyme. Although transgene expression differed among recipients, the level of enzyme was apparently sufficient to correct or ameliorate the histologic changes
consistent with PPCA deficiency
(Figures 2-6).
Correction of systemic pathology in PPCA / untreated
mice, mice transplanted with the MSCV-PPCA-marked BM had no systemic
manifestations of disease; they had a normal gross appearance, a shiny
fur, lack of diffuse edema, and inflammation of the eyelids, no tremor,
or ataxic movements up to 10 months after BMT. These features become
evident in PPCA/ mice starting at the age of
3 to 4 months.13,15,16 To assess the effect of
PPCA-expressing BM cells on organ morphology, we performed histologic
and immunohistochemical analyses of tissue sections. The combined use
of an anti-PPCA antibody, specific for the human PPCA, and an anti-GFP
antibody enabled us to discriminate between cells expressing the
transgene and cells that have internalized the corrective enzyme
(Figure 3). Transplanted mice were
analyzed at 1 to 10 months after treatment. Systemic correction was
observed in all MSCV-PPCA-transplanted animals, although the number of PPCA+ cells varied slightly in different mice, according to
the transduction efficiency and the repopulating capacity of
retrovirally marked BM cells. PPCA expression persisted long term,
indicating that sufficient numbers of HPCs were transduced. As
predicted by the levels of cathepsin A activity in various organs, we
detected high expression of PPCA in tissues of hematopoietic origin; in the spleen the distribution of PPCA-expressing cells was similar to
that observed in previous studies13,16 (Figure 3, -32
panel). This resulted in full reversal of the morphologic changes that remained apparent in untreated GS mice (Figure 2, BMT-PPCA and PPCA/). Clearance of storage material
occurred in the liver, both in Kupffer cells and in the hepatic
parenchyma (Figure 2, BMT-PPCA). Staining of adjacent sections with the
macrophage-specific anti-Mac-1 antibody (not shown) confirmed that the
BM-derived Kupffer cells were highly positive for PPCA (Figure 3,
-32 panel). In addition, hepatocytes displayed a PPCA-specific
punctate staining characteristic of lysosomes; this finding indicated
that PPCA was actively internalized (Figure 3, -32 panel). Foamy
histiocytes and vacuolated endothelial cells and hepatocytes persisted
in the untreated mice of similar age (Figure 2,
PPCA/). In the kidney, one of the most
severely affected organs in GS, PPCA-specific immunostaining was
observed throughout the renal parenchyma (Figure 3, -32 panel). This
feature was associated with complete resolution of lysosomal storage in
the proximal tubular and glomerular epithelia that instead was still
evident in PPCA/ mice (Figure 2, BMT-PPCA).
Strong immunostaining was also seen in the pulmonary alveolar
macrophages, the heart, the thymus, and the salivary glands (data not
shown). In all examined organs, the number of PPCA-expressing cells
exceeded that of GFP+ cells (Figure 3, -GFP panels) that
represented the population of transduced BM-derived cells that
repopulated the organs. These observations implied that efficient
cell-to-cell transfer of PPCA had occurred, resulting in the clearance
of lysosomal storage and correction of the systemic phenotype.
Amelioration of the pathologic changes in the CNS of recipient mice Regional distribution of CNS abnormalities in murine GS13,15 makes it difficult to accurately estimate whether isolated neuronal cells have been cleared of storage material. To ascertain the effects of transplanted, genetically corrected cells on the CNS phenotype, we performed histologic, immunochemical, and enzymatic analyses of the CNS at various time points after transplantation. Comparison of brains from mice that received MSCV-PPCA-transduced BM cells with those from PPCA/ mice revealed a significant
amelioration of the pathologic phenotype (Figure
4, BMT-PPCA and
PPCA/). In the regions most affected by PPCA
deficiency, including the cerebellar nuclei, the lateral geniculate
nuclei, and the amygdala, the amount of storage material appeared
reduced in recipients of MSCV-PPCA-transduced BM cells (Figure 4,
BMT-PPCA). The overall brain architecture was overtly improved in these
mice, likely because the endothelial cells and perivascular macrophages
were largely corrected. In accordance with this finding, immunoreactive PPCA in the brain was primarily restricted to leptomeningial, perivascular macrophages, and the vascular structure of the choroid plexus (Figure 5, -32). This
expression pattern coincided with that observed with -GFP antibody
(Figure 5, -GFP), although PPCA expression was more widely
distributed than GFP expression, and occasional neurons displaying a
clear punctate staining were observed only with the -32 antibody
(Figure 5).
The relatively small number of PPCA-expressing cells detected in neural tissues was in agreement with the low levels of cathepsin A activity measured in total brain lysates. However, given the overall improvement of brain morphology, it is apparent that only small amounts of enzyme are required for amelioration of brain pathology. Correction of the cerebellar defect in BM recipient mice A dramatic and progressive death of Purkinje cells occurs in the cerebellum of the GS mice, starting at the age of 3 to 4 months, and is one of the most overt consequences of this disease in the mice. Purkinje cells are lost in an anteroposterior and mediolateral fashion, the anterior lobes being the ones that are affected most and sooner. We have used this feature as a marker to monitor reduction in the neurologic damage after BMT. Serial sections of cerebella from mice that received MSCV-PPCA-transduced BM cells were compared with sections from wild-type and PPCA/ mice. The
appearance of Purkinje cells in PPCA-corrected mice was determined at 9 months after BMT by staining serial sections of the cerebella with an
antibody against PEP19.25 Purkinje cells were clearly more
numerous in treated mice than in age-matched PPCA mutant animals
(Figure 6). To quantify our observations, Purkinje cells were counted in these transplanted mice as well as in
one of the 3-month-treated group, and compared to 3- and 9-month-old
PPCA/ mice and age-matched controls.
Purkinje cells were counted at 2 levels: (1) in the paravermis at the
point where the lateral cerebellar nuclei first become obvious (medial)
and (2) in the hemisphere at the level of the dorsal cochlear nucleus
(hemisphere). In the medial region wild-type mice averaged 392 ± 19
Purkinje cells/section, whereas in the hemisphere the number was
362 ± 16. As expected, in the 3-month-old
PPCA/ animal only a small number of Purkinje
cells were lost: 24% in the medial and 21% in the hemisphere sections
(Figure 7). The total numbers were
practically identical in the 3-month-old-treated mouse, because the
variations in the different cerebellar regions were too small to be
detected. At this time point, there was also little variation in
Purkinje cell number between the anterior and posterior lobes of the
cerebellum. In contrast at 9 months, we observed a dramatic loss of
Purkinje cells in the PPCA/ mouse. In the
midline the total loss was 79%, but it was clearly more dramatic in
the anterior lobes of the cerebellum than in the posterior lobes, with
a loss of 93% and 61% of the cells, respectively. After BMT, the
rescue of Purkinje cells in the 9-month-old mice varied in different
cerebellar lobes, but the total number of cells was substantially
greater than that of age-matched PPCA/ mice
(Figure 7). In the medial cerebellum, the overall loss of Purkinje
cells in BMT recipient mice was 44% of controls. In the anterior lobes
of the BMT-treated medial cerebellum, 55% of the cells were missing,
whereas in the posterior lobes only 30% were. In the cerebellar
hemisphere, the overall loss of Purkinje cells in the BMT-treated mice
compared to the wild-type mice was 60%, with the anterior lobes
showing a 66% loss and the posterior lobes 51% loss of Purkinje
cells. These results support the notion that BMT of genetically
modified cells in GS mice delays the progressive loss of Purkinje cells
characteristic of the GS mice.
Transplantation of normal HPCs has been exploited for the
treatment of LSDs because BM progenitor cells can differentiate and
repopulate target organs, including the CNS, providing a permanent source of normal enzyme. The overall outcome of allogeneic and syngeneic BMT in patients and animal models has indicated that this
procedure is relatively effective in alleviating the systemic manifestations of the disease and in stabilizing bone lesions, especially if BMT is performed early in life.10,28-32
Correction, however, is often incomplete, suggesting that higher local
levels of gene expression may be required in some organs. Moreover,
diseases that have an early onset and involve predominantly the CNS
respond poorly to BMT, albeit that some variation in outcome has been observed among disease subtypes.10 The difficulty to
correct the CNS is attributed to the slow and incomplete engraftment of BM-derived cells into the adult brain33; it may also depend on the amount of enzyme secreted by normal cells, the extracellular stability of the enzyme,10 and the extent of uptake by
target cells. This conclusion is supported by our previous finding that complete systemic correction and partial amelioration of the brain pathology occur in GS mice that received transgenic BM in which cells
of the monocyte/macrophage lineage were modified to overexpress a PPCA
transgene.16 In these studies, neurologic abnormalities, including the loss of cerebellar function, were dramatically delayed when the transgenic mice were crossed into the
PPCA Building on these observations, we have now tested the hypothesis that
a similar or better outcome could be obtained in a gene therapy
setting, if sustained and long-term expression of the transgene could
be achieved. These studies allowed us to examine the feasibility of
such an approach for treatment of GS patients. Somatic gene therapy of
neurologic LSDs could be, in fact, the preferred treatment if
autologous HPCs could be engineered in vitro to constitutively express
and secrete high levels of the correcting enzyme. Early studies in
animal models have been disappointing with persistence of the lysosomal
defect and only negligible amelioration of the disease
phenotype.34 These results have been attributed to
ineffective transduction of HPCs, insufficient level or silencing of
transgene expression, immune depletion of the enzyme, or a combination
of these factors.35-37 However, some of these difficulties can now be circumvented by the use of improved viral vectors like the
one used in the treatment of We have established stable hematopoiesis in GS mice using
PPCA Despite the variability in levels of enzyme among recipients we can
conclude that sustained and long-term expression of PPCA, generated by
the MSCV retroviral cassette, undoubtedly contributed to the
prevention/correction of storage in the GS organs, including the CNS.
Most importantly, the extent of correction of the phenotype, including
the cerebellar defect, observed in BMT-treated GS mice is comparable to
that observed in crosses between PPCA Like the BMT approach, gene transfer into animal models of MPS I, MPS VII, Niemann-Pick disease, and metachromatic leukodystrophy has resulted in only partial correction of the enzyme deficiency in the brain although improvement in neurologic function could not be documented.48 Several features have been implicated in the poor response, including differences in disease type, the age of the animals at the time of transplantation, and the use of irradiation. To simulate a potential clinical intervention, we used total body irradiation (TBI), a conditioning modality important for engraftment in patients undergoing allogeneic HPC transplantation. TBI negatively affects neuronal development in infants. However, an unfavorable outcome due to TBI must be balanced with the ability of this procedure to disrupt transiently the integrity of the blood-brain barrier, and allow the entry of corrected cells into the CNS. Thus, it is possible that the high initial levels of enzyme that we achieved in this population and the early age at the time of treatment permitted the correction of a significant proportion of affected cells. Further studies in our model will be required to determine if sublethal doses of radiation allow a similar outcome. The combination of the high dose of cells, levels of HPC transduction,
and appropriate cellular expression of the corrective enzyme might have
played a crucial role in the histologic and functional correction of
the CNS pathology in treated mice. Given the devastation of the
cerebellar cortex in untreated PPCA
We are grateful to Gerard Grosveld for his continuous support, Tommaso Nastasi for his expert assistance in the preparation of the histology figures, Hongjun Wang for assistance in the preparation of the revised manuscript, and Charlette Hill for help in typing and editing the manuscript.
Submitted March 15, 2001; accepted December 4, 2001.
Supported by the Assisi Foundation of Memphis, National Institutes of Health grant RO1-DK 52025, the International Outreach Program at St Jude Children's Research Hospital (T.L.), the Cancer Center Support (CORE) grant CA 21765 from the National Cancer Institute, Phillip and Elizabeth Gross, and the American Lebanese Syrian Associated Charities (ALSAC). T.L., L.M., and M.d.P.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alessandra d'Azzo, Department of Genetics, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis, TN 38105; e-mail: sandra.dazzo{at}stjude.org.
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  C. Soulas, R. E. Donahue, C. E. Dunbar, D. A. Persons, X. Alvarez, and K. C. Williams Genetically Modified CD34+ Hematopoietic Stem Cells Contribute to Turnover of Brain Perivascular Macrophages in Long-Term Repopulated Primates Am. J. Pathol., May 1, 2009; 174(5): 1808 - 1817. [Abstract] [Full Text] [PDF]
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 Y. Oheda, M. Kotani, M. Murata, H. Sakuraba, Y. Kadota, Y. Tatano, J. Kuwahara, and K. Itoh Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and galactosialidosis by normal gene transfer and enzyme replacement Glycobiology, April 1, 2006; 16(4): 271 - 280. [Abstract] [Full Text] [PDF]
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 R. Sano, A. Tessitore, A. Ingrassia, and A. d'Azzo Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology Blood, October 1, 2005; 106(7): 2259 - 2268. [Abstract] [Full Text] [PDF]
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 M. Nour, A. B. Quiambao, M. R. Al-Ubaidi, and M. I. Naash Absence of Functional and Structural Abnormalities Associated with Expression of EGFP in the Retina Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 15 - 22. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-galactosidase
and N-acetyl-
/
