Current Pharmaceutical Biotechnology
Substrate reduction therapies for mucopolysaccharidoses
Joanna Jakóbkiewicz-Banecka1, Ewa Piotrowska1, Magdalena Gabig-Cimińska2,
Elżbieta Borysiewicz1,3, Monika Słomińska-Wojewódzka1, Magdalena Narajczyk1,
Alicja Węgrzyn2 and Grzegorz Węgrzyn1,*
1
Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland
2
Laboratory of Molecular Biology (affiliated with the University of Gdańsk), Instituite of
Biochemistry and Biophysics, Polish Academy of Sciences, Kładki 24, 80-822 Gdańsk, Poland
3
Department of Neurobiology and Anatomy, West Virginia University School of Medicine,
Morgantown, WV 26506-9128, USA
* Corresponding author:
Prof. Grzegorz Węgrzyn
Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland
Tel. +48 58 523 6308; Fax: +48 58 523 5501
e-mail: wegrzyn@biotech.univ.gda.pl
Running title: Substrate reduction therapies
1
ABSTRACT
Mucopolysaccharidoses (MPS) are inherited metabolic disorders, caused by mutations leading
to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs) in
lysosomes. Due to their impaired degradation, GAGs accumulate in cells of patients, which
results in dysfunction of tissues and organs, including the heart, respiratory system, bones,
joints and central nervous system. Depending on the kind of deficient enzyme, 11 types and
subtypes of MPS are currently recognized. Although enzyme replacement therapy has been
developed for 3 types of MPS (types I, II and VI), this treatment was found to be effective
only in management of somatic symptoms. Since all MPS types except IVA, IVB and VI are
characterized by various problems with functioning of the central nervous system (CNS), a
search for effective treatment of this system is highly desirable. Recent discoveries suggested
that substrate reduction therapy may be an efficient method for treatment of MPS patients,
including their CNS. In this review, different variants of this therapy will be discussed in the
light of recently published reports.
Key words: mucopolysaccharidoses; lysosomal storage diseases; substrate reduction therapy;
gene expression-targeted isoflavone therapy; siRNA; shRNA
2
INTRODUCTION
Treatment of neurodegenerative diseases is a challenge for current pharmacology and
biotechnology [1-5]. The complicated structure of brain and problems with delivery of certain
drugs, especially those consisting of large biomolecules, through the blood-brain-barrier
(BBB) make the problem especially difficult. On the other hand, neurodegenerative diseases
are relatively frequent and often very severe, with high morbidity and mortality, thus, being
considered as a major medical and social problem.
Some neurodegenerative diseases are inherited, which adds another difficulty to their
treatment as little, if any, prophylactic procedures can be used to prevent their development.
Although gene therapy might be a potential effective treatment for such disorders [6], the
problem is that this kind of therapy is still under development and despite many tests on
animal models and still ongoing clinical trials, there is no gene therapy procedure officially
approved as a treatment for neurodegenerative disease. Among inherited neurodegenerative
disorders, there is a large group of lysosomal storage diseases (LSD), caused by dysfunction
of one or more lysosomal enzymes, characterized by neuronopathic symptoms [7]. Since
molecular mechanisms of LSD are rather well understood (relative to many other diseases of
this nature), and as they were among the first genetic diseases for which effective – at least to
some extent - treatment became possible, these disorders may be considered as models in
inherited metabolic diseases, including its neurodegenerative component.
The treatment for LSD, mentioned above, is enzyme replacement therapy (ERT) [8].
This therapy is based on intravenous infusion of a recombinant human enzyme which is
lacking or deficient in patient’s cells. Due to specific signal label attached to the protein core
of the enzyme, it can be recognized by cellular receptors of lysosomal proteins and
transported to lysosomes inside cells. However, despite such a recombinant enzyme can be
3
delivered to most tissues and organs, it cannot cross BBB, and thus, ERT is not effective in
treatment of neurological symptoms.
MUCOPOLYSACCHARIDOSES
Mucopolysaccharidoses (MPS) belong to LSD and are caused by mutations in genes
coding for enzymes involved in degradation of glycosaminoglycans (GAGs) (formerly called
mucopolysaccharides) [9]. Impaired hydrolysis of GAGs leads to continuous accumulation
and storage of these compounds in cells of patients, which result in a progressive damage of
the affected tissues, including the heart, respiratory system, bones, joints and CNS (in most
MPS types and subtypes). MPS are usually fatal diseases, with average expected life span of
one or two decades, though patients with milder forms can survive into adulthood [9].
There are 11 known types and subtypes of MPS, depending on the nature of lacking or
defective enzyme and the kind(s) of stored GAG(s). Among those 11 forms of MPS,
neurodegeneration occurs in most of them, with only three (types IVA, IVB and VI) definitely
free of neuronopathy (Table 1). Until now, only one patient suffering from MPS IX has been
described [10], and although no neurological symptoms were found, this case in nonconclusive on whether MPS IX is a neuronopathic or non-neuronopathic disease.
A characteristic feature of each MPS type is a huge variability in the spectrum and
severity of symptoms [9]. Prediction of severity and clinical progression of MPS is difficult,
or even impossible, despite biochemical and genetic data may be available [11]. Recent
studies suggested that considering two or more parameters may give significantly better
results than attempting to make conclusion based on a single biomarker [12]. Nevertheless,
although the variability of MPS makes serious problems for clinicians who must decide about
choosing potentially optimal management and symptomatic treatment of patients, this feature
4
provides a tool for studying molecular mechanisms of the disease and developing novel
therapeutic strategies [11]. Thus, MPS can be considered as good models in biotechnological
studies on development of new pharmaceuticals for treatment of neurodegenerative genetic
diseases.
Currently, bone marrow or hematopoietic stem cells’ transplantations (with still
unproved efficacy) and ERT are the only officially approved treatments of MPS. ERT is used
in a clinical practice for only 3 MPS types, namely MPS I, II and VI [8, 13]. As mentioned
above, neurological symptoms developed due to GAG accumulation in CNS cannot be
managed by ERT owing to an inefficient delivery of proteins through BBB [13]. Although
gene therapy is a hope for MPS patients, it still remains a treatment under development rather
than a real therapy [14]. Therefore, there is still a need for alternative therapies, which could
be helpful for patients suffering from MPS, especially from neuronopathic forms. Moreover,
since advances in biochemistry and genetics have resulted in understanding molecular bases
of MPS, with subsequent development of specific treatment regimens for some of these
diseases (ERT for MPS I, II and VI), it is believed that experience gained in MPS by the
development, evaluation and integration of treatment regimens into healthcare may be
instructive for other genetic disorders [15].
DEVELOPMENT OF SUBSTRATE REDUCTION THERAPIES FOR
MUCOPOLYSACCHARIDOSES
Since neither bone marrow transplantation nor enzyme replacement therapy can be
effective in treatment of neurological symptoms of MPS, especially severe ones, development
of alternative therapies became necessary, especially for MPS types in which neuronopathic
forms predominate, like Sanfilippo disease (Table 1). Several approaches has been tested,
5
with some achieving the phase of clinical trials, however, such trials with glucosamine (which
was expected to alleviate symptoms of MPS III due to its similarity to the terminal
carbohydrate moiety present in partially degraded heparan sulfate molecules that are stored in
Sanfilippo cells) and miglustat (which should inhibit synthesis of gangliosides, compounds
that are secondary products of GAG storage [16]) were not successful. Therefore, there is still
a need for effective method to treat neuronopathic forms of MPS (for reviews see [17-19]).
One of potential therapeutic method for LSD is substrate reduction therapy (SRT),
called also sometimes substrate deprivation therapy (SDT) [19]. This kind of therapy is based
on an assumption that inhibition of synthesis of compounds that cannot be efficiently
degraded, may facilitate an establishment of a new balance between their production and
degradation, already lost due to a defect in a specific hydrolase [20]. Reports of recent years
indicated that this approach may be of surprisingly high efficacy in not only reducing
synthesis of GAGs in MPS cells, but also in decreasing lysosomal storage. Results of studies
on cell cultures and animal models were very encouraging, and recent pilot clinical studies
confirmed that this method may be effective in improvement of at least some clinical
parameters of patients suffering from neuronopathic MPS, including cognitive functions. In
following chapters we will summarize and discuss these results in the light of possible
development of the treatment that can be approved as a method for MPS therapy.
Rhodamine B
Rhodamine B ([9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]diethylammonium chloride) is a synthetic chemical, commonly used in biological studies as a
staining fluorescent dye. It has been demonstrated that this compound reduced GAG synthesis
in cultures of MPS IIIA and MPS VI cells [21]. Moreover, addition of rhodamine B into
6
cultures of MPS IIIA and MPS VI fibroblasts resulted in a significant decrease in lysosomal
storage of GAGs [21]. MPS IIIA mice were treated with rhodamine B at the dose of 1 mg/ml,
and GAG levels were reduced relative to untreated animals. Particularly, liver size, total GAG
content, and lysosomal GAG was reduced as was urinary GAG excretion. It is of special
interest that lysosomal GAG content in the brain was also significantly reduced by this
treatment, which was perhaps possible because of a potential of rhodamine B to cross BBB
[21]. These results demonstrated that SRT is even more efficient in treatment of MPS
symptoms that it was expected earlier. Namely, it was supposed that inhibition of the
substrate production might slow down or, at best, halt the progression of the disease, rather
than reverse the symptoms.
Of outstanding importance are studies on MPS IIIA mice treated with rhodamine B, in
which behavioral tests were performed, and spatial learning and memory were assessed [22].
Contrary to control MPS IIIA mice, the rhodamine B-treated MPS IIIA mice improved
performance towards normal animals [22]. These results provided the first evidence for
beneficial effect on CNS function in an MPS disorder due to the use of SRT.
Although experiments with rhodamine B provided important support for efficacy of
SRT in MPS, there are serious problems which preclude the use of this compound as a drug
for humans. Firstly, rhodamine B appears to be a non-specific inhibitor of GAG synthesis
[21], and in fact, a mechanism for its action is unknown (Fig. 1). Secondly, and perhaps more
importantly, rhodamine B is supposed to be toxic for humans [21]. It is considered harmful to
humans if swallowed, and may cause irritation of skin, eyes and respiratory tract (see, for
example: http://www.jtbaker.com/msds/englishhtml/r5400.htm). Therefore, despite very
promising results of studies on cell cultures and mice, it is unlikely that rhodamine B can be
used as a drug for MPS treatment.
7
Gene expression-targeted isoflavone therapy
Simultaneously with studies on rhodamine B, research on inhibition of GAG synthesis
by another compound, genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4one) was conducted. Genistein is a natural isoflavone occurring in many plants, and known to
possess various biological activities, ranging from phytoestrogenic to antioxidative actions
[23, 24]. When added to cultures of fibroblast of MPS I, MPS II, MPS IIIA and MPS IIIB
patients, this compound, at concentrations between 10 and 30 M, inhibited GAG synthesis
and reduced lysosomal GAG storage [25].
Contrary to rhodamine B, it was possible to learn about the mechanism of genisteinmediated inhibition of GAG synthesis. An excess of epidermal growth factor (EGF) abolished
genistein-mediated impairment of GAG synthesis in cultured fibroblasts, while increased
concentrations of genistein partially restored this negative regulation [26]. Moreover,
genistein affected EGF receptor-catalyzed phosphorylation efficiency [26]. EGF is a ligand,
whose binding by its specific receptor (EGF receptor) triggers a signal transduction pathway
resulting in activation of expression of certain genes [27], apparently including genes coding
for enzymes required for GAG synthesis. Therefore, the potential therapy based on the use of
genistein (an isoflavone) as an inhibitor of the EGF-mediated signal transduction pathway,
has been named ‘gene expression-targeted isoflavone therapy’ (GET IT) [25, 26]. In this light,
effects of the action of genistein on MPS cells are compatible with results of recent studies,
indicating that efficiency of GAG synthesis may significantly influence severity of MPS
diseases [28]. The mode of genistein action as an inhibitor of GAG synthesis is summarized
in Fig. 1.
Similarly to rhodamin B, genistein caused a marked improvement in treated mice
suffering from Sanfilippo disease (in these experiments, MPS IIIB mice were used) [29].
8
Importantly, no adverse effects related to genistein administration were observed, despite the
doses used were from 5 mg/kg to as high as 160 mg/kg.
Efficacy in pre-clinical studies and a lack of toxicity of genistein, confirmed by results
on the MPS IIIB mouse model [29] and deduced on the basis of a large body of previously
published data [30], encouraged researchers and clinicians to perform a pilot clinical study on
the use of GET IT in treatment of patients suffering from Sanfilippo disease. In this openlabel study, 10 patients diagnosed as MPS IIIA or MPS IIIB were treated for 12 months with
a genistein-rich soy isoflavone extract at the dose corresponding to 5 mg genistein per 1 kg of
body weight daily [31]. This treatment, in which no adverse effects were noted, resulted in a
statistically significant improvement in all tested parameters in this group of patients [31].
Namely, urinary GAG levels were reduced, hair morphology (suggested to be a useful
parameter in monitoring efficacy of treatment of patients suffering from various MPS types
[32-34]) improved, and patients got higher scores in the psychological test by which cognitive
functions could be assessed [31].
The improvement in brain functions (assessed by estimation of cognitive abilities) of
MPS III patients after treatment with GET IT appears to be of special importance, considering
the nature of symptoms of Sanfilippo disease. In fact, the paper describing these results [31]
was the first report demonstrating an improvement of MPS III patients after pharmacological
treatment. Moreover, it indicated that neurological functions, already lost due to
neurodegeneration in the course of a genetic disease, can be restored (at least partially) after
pharmacological treatment.
It is interesting to ask whether action of genistein as an inhibitor of the EGF-mediated
signal transduction pathway is the only mechanism causing efficacy of GET IT? Beside
storage of GAGs in cell lysosomes and secondary storage of gangliosides (mentioned above),
oxidative stress, inflammation, cytotoxicity and apoptosis were postulated to be involved in
9
the mechanism of neurodegeneration in MPS [35-37]. In addition, accumulation of
hyperphosphorylated tau protein (P-tau), which forms aggregates characteristic for Alzheimer
disease, has been detected in brains of MPS IIIB mice [38]. Considering these facts, it is of
interest to note that genistein may be effective in prevention of the devastating processes in
brain or amelioration of the function of affected brain. Namely, genistein could attenuate
oxidative stress in brain [39], and revealed neuroprotective effect against beta amyloidinduced neurotoxicity [40, 41]. Moreover, genistein inhibited apoptosis in primary neuronal
cell cultures [42] and prevented Alzheimer's disease-associated inflammation [43]. General
neuroprotective effects of genistein were also described [44]. Therefore, one might speculate
that various neuroprotective activities of this isoflavone may contribute to the mechanism of
efficacy of GET IT. It was also demonstrated that genistein can enhance expression of
synaptophysin [45]. This can suggest a genistein-mediated stimulation of synapse formation,
which might serve as a partial explanation of the phenomenon of gaining cognitive functions
by MPS III patients subjected to GET IT [31] that are otherwise continuously deteriorating.
RNAi-mediated silencing of genes involved in GAG synthesis
RNA interference (RNAi) is a process regulating gene expression by sequencedirected silencing of translation due to degradation of specific mRNA or translation inhibition
[46]. Since impairment of GAG synthesis appeared effective in storage reduction in MPS
cells (see preceding sections), the idea appeared that silencing of specific genes coding for
enzymes involved in GAG production may be a precise method for substrate reduction
therapy for MPS. Therefore, two research groups developed procedures to impair expression
of such genes, though various genes were chosen and different techniques were employed.
10
One group has used shRNA molecules specific to EXTL2 and EXTL3 genes [47]. A
significantly reduced expression of endogenous target genes was observed, as was a decreased
GAG synthesis. Moreover, lysosomal GAG levels were reduced in MPS I and MPS IIIA
fibroblasts treated with EXTL2- and EXTL3-specific shRNAs [47].
The second group employed siRNA oligonucleotides to reduce mRNA levels of four
genes, XYLT1, XYLT2, GALTI and GALTII, whose products are required for GAG synthesis
[48]. Following transfection of MPS IIIA fibroblasts, a significant decrease in levels of
corresponding transcripts was observed. This was accompanied by a decrease in levels of
proteins encoded by these genes. Efficiency of GAG production in the fibroblasts was
considerably reduced after treatment of these cells with siRNA [48].
The results reported by both groups [47, 48] indicated that impairment of expression
of particular genes coding for enzymes required for GAG synthesis may be effective in
reduction of lysosomal storage in various MPS types. Moreover, they support the conclusion
that SRT can be a method suitable for treatment of patients suffering from MPS. Although
different genes were silenced in experiments performed by certain groups (Fig. 2), and thus,
GAG synthesis was inhibited at various stages of the synthesis pathway, efficiency of this
inhibition was comparable in all cases, and corresponded to efficiency of reduction of the
storage material [47, 48].
The main problem with the treatment of neuronopathic MSP types is delivery of the
specific drugs to CNS, especially to brain, and this problem concerns also potential treatments
with the use of the RNAi-based methods. Moreover, one must also consider a low stability of
RNA in vivo. Nevertheless, recent achievements in the field of the use of RNAi in medicine
are encouraging, and give a hope that effective treatment of neurological diseases with RNAibased drugs may be introduced relatively soon [49, 50].
11
CONCLUDING REMARKS
Although various kinds of therapeutic concepts have been considered to treat
neuronopathic forms of MPS, the therapy based on impairment of synthesis of compounds
that cannot be degraded efficiently in cells, called SRT, was the first one that showed some
efficacy in clinical studies. This can be an indication that SRT might be a potentially effective
treatment of other neurodegenerative diseases caused by storage of certain compounds.
Undoubtedly, further optimization of drug dosage and delivery is required to make SRT for
MPS even more effective, however, first positive effects of clinical studies strongly suggest
that this kind of treatment is a hope for patients suffering from metabolic brain diseases. We
believe that combination of SRT with other therapeutic procedures, like ERT, might give
optimal effects, significantly increasing quality of life of these patients.
ACKNOWLEDGMENTS
This work was supported by Ministry of Sciences and Higher Education of Poland (project
grants no. N302 046 32/3603 to G.W. and 3631/B/P01/2007/33 to J.J.-B.), and was operated
within the Foundation for Polish Science Team Programme co-financed by the EU European
Regional Development Fund (grant no. TEAM/2008-2/7 to G.W.).
12
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20
Table 1. Mucopolysaccharidoses (MPS), stored GAGs, deficient enzymes and neurological
symptoms
MPS type
(syndrome name)
MPS I (Hurler
disease - MPS I-H,
Scheie disease –
MPS I-S, HurlerScheie disease –
MPS I-H/S)
MPS II (Hunter
disease)
MPS IIIA
(Sanfilippo disease
subtype A)
MPS IIIB
(Sanfilippo disease
subtype B)
MPS IIIC
(Sanfilippo disease
subtype C)
MPS IIID
(Sanfilippo disease
subtype D)
MPS IVA
(Morquio disease
subtype A)
MPS IVB
(Morquio disease
subtype B)
MPS VI
(Maroteaux-Lamy
disease)
MPS VII (Sly
disease)
MPS IX
Main GAGs
stored in cellsa
DS, HS
Deficient enzyme
Neurological symptoms
-L-iduronidase
Severe (in the MPS I-H clinical
subtype), very mild to mild (in
MPS I-H/S) or none (in MPS IS)
DS, HS
Iduronate sulfatase
Severe or mild (rarely none)
HS
heparan N-sulfatase
Severe
HS
-N-acetylglucosaminidase
Severe
HS
acetyl-CoA:glycosaminide
acetyltransferase
N-acetylglucosamine
6-sulfatase
Severe
KS
N-acetylgalactosaminide 6-sulfatase
None
KS
-galactosidase
None
DS
N-acetylgalactosamine 4-sulfatase
None
DS, HS
-glucuronidase
Severe or mild (rarely none)
Hyaluronan
Hyaluronidase
Noneb
HS
Severe
Abbreviations: DS – dermatan sulfate, HS – heparan sulfate, KS – keratan sulfate
Only one patient has been described to date, thus, this case is non-conclusive for occurrence
of neurological symptoms in this disease.
a
b
21
FIGURE LEGENDS
Figure 1. A scheme for expression of genes coding for enzymes involved in GAG synthesis,
with stages inhibited by various methods of SRT indicated by blunt-ended lines. Mechanism
of the rhodamine B-mediated inhibition is not known. Competitive inhibition of enzymes by
monosaccharide analogs (dashed line) appears unlikely to be used as a therapeutic procedure
due to potential side effects caused by blocking of other biochemical pathways, in which these
monosaccharides are involved.
Figure 2. A scheme for syntheses of heparan sulfate and dermatan sulfate (two kinds of
GAGs, stored in most MPS types, except MPS IV and IX) with marked enzymes catalyzing
particular reactions. Monosaccharides used for GAG synthesis are indicated by symbols
explained in the figure. Silencing of expression of particular genes, coding for corresponding
enzymes, by either siRNA (according to Dziedzic et al. [48]) or shRNA (according to
Kaidonis et al. [47]), is indicated.
22
Figure 1.
Figure 2.
23