Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders

ACCEPTED ARTICLE PREVIEW

Accepted Article Preview: Published ahead of advance online publication PHARMACOLOGICAL CHAPERONE THERAPY: PRECLINICAL DEVELOPMENT, CLINICAL TRANSLATION, AND PROSPECTS FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDERS

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GiancarloParenti, Generoso Andria, Kenneth J. Valenzano

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Cite this article as: GiancarloParenti, Generoso Andria, Kenneth J. Valenzano, PPHARMACOLOGICAL CHAPERONE THERAPY: PRECLINICAL DEVELOPMENT, CLINICAL TRANSLATION, AND PROSPECTS FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDERS, Molecular Therapy accepted article preview online 16 April 2015; doi:10.1038/mt.2015.62

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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 19 February 2015; accepted 01 April 2015 ; Accepted article preview online 16 April 2015

© 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW PHARMACOLOGICAL CHAPERONE THERAPY: PRECLINICAL DEVELOPMENT, CLINICAL TRANSLATION, AND PROSPECTS FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDERS

Authors: Giancarlo Parenti1,2, Generoso Andria1, Kenneth J. Valenzano3 1

Department of Translational Medical Sciences, Section of Pediatrics, Federico II University,

Via S. Pansini 5 Naples, Italy Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, Pozzuoli, Italy

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Amicus Therapeutics, 1 Cedar Brook Drive, Cranbury, NJ 08512, USA

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Giancarlo Parenti ([email protected])

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Correspondence should be addressed to

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Short title: Chaperone therapy for lysosomal storage disorders

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Federico II University,

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Department of Translational Medical Sciences, Section of Pediatrics,

Via S. Pansini 5

80131 Naples, Italy

Fax: +39 081 746 3116; email: [email protected] and Telethon Institute of Genetics and Medicine, Viale Campi Flegrei 34, 80078 Pozzuoli, Italy email: [email protected] 1 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Abstract Lysosomal storage disorders (LSDs) are a group of inborn metabolic diseases caused by mutations in genes that encode proteins involved in different lysosomal functions, in most instances acidic hydrolases. Different therapeutic approaches have been developed to treat these disorders. Pharmacological chaperone therapy (PCT) is an emerging approach based on smallmolecule ligands that selectively bind and stabilize mutant enzymes, increase their cellular levels, and improve lysosomal trafficking and activity. Compared to other approaches, PCT

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shows advantages, particularly in terms of oral administration, broad biodistribution, and positive

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impact on patients’ quality of life. After preclinical in vitro and in vivo studies, PCT is now being translated in the first clinical trials, either as monotherapy or in combination with enzyme

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replacement therapy, for some of the most prevalent LSDs. For some LSDs, the results of the

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first clinical trials are encouraging and warrant further development. Future research in the field

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of PCT will be directed towards the identification of novel chaperones, including new allosteric drugs, and the exploitation of synergies between chaperone treatment and other therapeutic

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approaches.

Key words: pharmacological chaperone therapy, lysosomal storage disorders, allosteric chaperone, active site-directed chaperone, combination therapy

2 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Lysosomal Storage Disorders Lysosomal storage disorders (LSDs) represent a heterogeneous group of over 50 distinct diseases, each of which results from functional deficiency of a particular lysosomal protein. For the majority of LSDs, the defective protein is a soluble acidic hydrolase, while several others are caused by deficiency of integral membrane, activator, transporter, or non-lysosomal proteins that are necessary for lysosomal function. Deficiency of a lysosomal function invariably leads to the accumulation of a wide range of complex substrates, which may include various

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glycosphingolipids, glycosaminoglycans, glycogen, oligosaccharides, cholesterol, peptides,

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and/or glycoproteins,1,2 and in secondary impairment of lysosome-related pathways.3 Storage of different substrates in multiple organs and systems results in the variable association of visceral,

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ocular, hematologic, skeletal, and neurological manifestations. Severity of clinical

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manifestations, age at onset, and disease course often vary among individuals with the same

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LSD, resulting in broad clinical presentation. In general, LSDs substantially impact patients’ health, quality of life, life expectancy, and physical and intellectual performance. While each

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LSD is quite rare, collectively they affect a large number of individuals, with an estimated incidence rate of 1:5000 to 1:7000 live births.4 For their impact on patients’ health and for their cumulative frequency, LSDs represent a heavy burden in terms of public health and economical costs. Treatment of LSDs Even though LSDs are rare, their biological and clinical interest is high. These diseases represent models to understand lysosomal function, and its role in cellular biology. Furthermore, over the past 25 years intensive and continuous advancements have been made to develop therapies that are specifically aimed at correcting the metabolic defect(s) of LSDs. 5 3 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

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The majority of these therapeutic approaches are directed towards increasing the cellular activity or level of the defective enzyme or protein, with the ultimate goal of lowering the accumulated substrate in key cell types and tissues. Given that the underlying cause of most LSDs is deficiency of a particular enzyme activity, the primary therapeutic approach that has been most successful and broadly reached to date is enzyme replacement therapy (ERT). ERT is based on the periodic intravenous administration of a manufactured enzyme that can be taken up into cells,

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be delivered to lysosomes, and reduce substrate storage.6 To date, seven LSDs have marketed

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ERT products, and in some cases multiple products exist for a single LSD.

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Similar to ERT, normal enzyme also may be provided as a precursor that is secreted into the circulation by allograft of transplanted cells (hematopoietic stem cell

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transplantation, HSCT) ,7 or by a patient’s own engineered cells.8 Similarly, the gene

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mutation may potentially be corrected by delivering a wild-type copy that will direct the synthesis of the normal enzyme in the patient’s cells.9 Alternative strategies also exist,

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and are directed towards reducing the synthesis of substrates (i.e., substrate reduction therapy, for which two drugs currently exist),10 by enhancing clearance of substrates from cells and tissues, or by manipulating specific cellular pathways (such as those involved in vesicle trafficking) .11 The focus of this paper however, is pharmacological chaperones, small molecules that can selectively bind and stabilize mutant enzymes, protecting them from premature denaturation and degradation, and ultimately increasing their lysosomal levels and activity.12,13,14 Recently, pharmacological chaperone therapy (PCT) for a number of LSDs has been evaluated in the clinic, with some molecules showing therapeutic promise. 4 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW The Concept of PCT The balance between protein synthesis, folding, and degradation is a closely monitored and highly dynamic process that is critical to maintain cellular protein homeostasis, i.e., proteostasis (FIGURE 1).15,16 Cells have evolved complex quality control systems that function in various organelles, including the endoplasmic reticulum (ER), to aid in these processes.16,17 These mechanisms rely on multiple classes of proteinaceous molecular chaperones and folding factors (e.g., heat-shock proteins) that recognize and co-translationally interact with various partially

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folded, aggregation-prone structural motifs of nascent proteins during synthesis, such as exposed

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hydrophobic regions, regions of unstructured polypeptide sequence, or unpaired cysteine residues, to distinguish stable, native conformations from unstable, misfolded conformations16.

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Upon recognition and binding to the nascent polypeptide, molecular chaperones can stabilize

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protein conformation, inhibit premature misfolding, and prevent aggregation.18,19,20 Only those

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proteins that are correctly folded and stable can exit the ER efficiently and traffic to the lysosome. If folding of the nascent protein fails despite the action of molecular chaperones, the

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protein may be recognized by the ER quality control system as aberrant and targeted for degradation either by the lysosome or the proteasome.21,22 For the latter, ER-associated degradation involves polyubiquination, translocation to the cytosol, and proteasomal degradation.

In the case of LSDs, the ER quality control often recognizes mutant forms of lysosomal enzymes that retain catalytic activity, or that have only modestly compromised function, due to slight modifications in their stability or conformation. This recognition may prevent trafficking through the secretory pathway, and result in loss of function due to premature degradation or ER 5 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW aggregation. Based on an evolving understanding of these mechanisms, several small molecule approaches to correct deficiencies that result from mutations in lysosomal enzymes have emerged over the last 15 years.16 One of the more promising and advanced approaches utilizes pharmacological chaperones: small-molecule ligands that selectively bind and stabilize otherwise unstable enzymes to increase total cellular levels and improve lysosomal trafficking and activity (FIGURE 2). Lysosomal enzyme synthesis occurs in the neutral pH environment of the ER. As these enzymes

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are acidic hydrolases, many tend to be thermodynamically less stable at neutral pH compared to

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the low-pH environment of lysosomes.23 For some mutant lysosomal enzymes, this thermodynamic instability can be exacerbated, with consequently even less of the properly

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folded enzyme able to exit the ER. In this context, pharmacological chaperones selectively bind

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and stabilize a specific target enzyme, resulting in increased total cellular levels, and passage

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through the quality control mechanisms of the ER, with subsequent delivery to lysosomes.12,13

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Why Use PCT to Treat LSDs?

For a number of reasons, LSDs can be considered excellent candidates for PCT. LSDs are often caused by mutations that are associated with protein misfolding. While many types of mutations have been identified in LSDs, including large deletions, insertions, premature stop codons, and splicing mutations, missense mutations tend to be more common.13 In most cases, missense mutations occur outside the enzyme’s active site and have negative effects on protein folding efficiency, thermodynamic stability, and lysosomal trafficking, although the mutant enzymes retain their catalytic properties.16 This concept has been characterized in detail for several LSDs. For example, Gaucher disease, generally thought to be the most prevalent 6 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW LSD, is caused by mutations in the GBA1 gene that encodes glucocerebrosidase (GCase; EC 3.2.1.45),24 and is characterized by progressive accumulation of glucosylceramide primarily within macrophages of the liver, bone marrow, and spleen. Among the over 200 mutations identified in GBA1, the two most prevalent missense mutations are N370S and L444P,24 retaining approximately 30% and 10-12% residual cellular activity, respectively.25 These mutations result in deleterious changes in the three-dimensional structure, and in variable levels

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of ER retention and ER-associated degradation.26

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Evidence that mutations in the genes encoding different lysosomal enzymes result in protein misfolding, retention in the ER/Golgi, degradation, lack of appropriate protein processing, and/or

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defective transport to lysosomes has also been reported for many other LSDs.27 Since the

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mutations that cause misfolding are relatively prevalent in some LSDs, like Gaucher disease and

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Fabry disease, PCT has the potential to be a suitable strategy for the treatment of a substantial

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fraction of affected patients.

Minimal increases in activity may be sufficient to positively impact phenotype. For most LSDs, it has been speculated that substrate storage occurs if residual enzyme activity falls below a certain threshold (FIGURE 3), and that above a certain level, accumulation of clinically relevant quantities of substrate would require a duration that exceeds the human lifespan.28 It has been assumed that a threshold activity of approximately 10% is sufficient to prevent storage in several LSDs, with restoration of 3% to 5% activity often cited as sufficient to slow disease progress.29,30,31 This suggests that enhancement of 7 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW the residual cellular activity by PCT may attenuate disease progression, and translate into benefit for patients. However, the residual activity known to be associated with attenuated phenotypes and required to prevent massive storage seems to vary among different disorders, and may depend, in part, on various factors such as the severity of the enzymatic defect, , the rate of substrate accumulation/turnover, the stage of disease progression, sex, etc. For example, while 1% – 2% of normal GCase and alphaiduronidase activity have been reported for some mild cases of Gaucher disease and

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mucopolysaccharidosis I, respectively, higher activities (6%) have been reported in late-

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onset GM1 gangliosidosis13.

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Existing therapies for LSDs show major limitations. ERT is currently considered the standard of

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care for several LSDs. While some of the underlying pathologies and affected organ systems are

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treated well by ERT, others are not. This is often due to poor tissue access, as well as inefficient cellular uptake and lysosomal delivery. In particular, the ability to target the central nervous

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system has not been achieved, given the inability of large proteins to cross the blood-brain barrier.32,33 Furthermore, most of the current ERTs are immunogenic, eliciting immune responses that can limit tolerability and efficacy.34,35,36,37 In addition, ERT requires lifelong intravenous infusion, with frequent hospital admissions, need for central venous devices (and related risk of infections), and high costs.38 The other therapeutic approaches mentioned above (e.g., substrate reduction therapy, HSCT, gene therapy) are either restricted to a few LSDs, or are in early stages of clinical development.

8 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW In principle, PCT has the potential to address at least some of the limitations of existing therapies. Pharmacological chaperones are small molecules with published data indicating, as a class, good oral bioavailability, broad tissue distribution to key cell types and tissues including the brain, and the ability to diffuse across membranes, achieving therapeutic concentrations in specific cellular compartments. Enhancement of enzyme activity by PCT also may lead to sustained and stable enzyme levels, more closely mimicking the natural production of these endogenous enzymes as compared to weekly

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or biweekly ERT administration that leads to intermittent and fluctuating cellular activity.

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In addition, unlike the manufactured enzymes used for ERT, pharmacological chaperones are non-immunogenic, and would not be expected to have tolerability

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issues similar to those described for a number of different ERTs. In fact, the safety

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appear to be acceptable.

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profiles of some pharmacological chaperones have been evaluated clinically, and

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Preclinical Development of PCT for LSDs Since 1999, PCT has been evaluated in preclinical studies for a number of LSDs. TABLE 1 provides a list of selected chaperones that have shown the greatest potential for clinical studies. The first studies on PCT in an LSD were done for Fabry disease, an X-linked disorder caused by mutations in the GLA gene that encodes α-galactosidase A (α-Gal A; EC 3.2.1.22).39 Deficiency of α-Gal A activity results in accumulation of glycosphingolipids, primarily globotriaosylceramide (GL-3) and globotriaosylsphingosine (lyso-Gb3), in various organs and tissues.39 A natural substrate mimetic, 1-deoxygalactonojirimycin (DGJ, AT1001, migalastat), was described as a pharmacological chaperone for α-Gal A.40,41 DGJ binds reversibly and 9 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW selectively to the active site of α-Gal A with high affinity,42 and can increase in vitro the cellular activity of different missense mutant forms of α-Gal A with concomitant reductions in GL3.43,44,45 In vivo studies using a transgenic mouse model that expresses the human R301Q transgene on a Gla knock-out background (hR301Q α-Gal A Tg/KO mice) showed increased αGal A activity and reduced GL-3 levels in various disease-relevant tissues following oral

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administration of DGJ.46

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PCT also has been proposed as a potential therapy for Gaucher disease.47,48 A wide variety of

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compounds that increase the cellular activity of various mutant forms of GCase in cell lines

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derived from Gaucher patients have been evaluated. These molecules can be categorized as carbohydrate mimetics (iminosugars, azasugars, carbasugars), or non-carbohydrate compounds

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identified by high-throughput screening initiatives.13,14 From this collection, two appeared to be

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particularly promising and have advanced through preclinical studies and into early-stage clinical

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development, namely isofagomine (IFG, AT2101, afegostat tartrate) and Ambroxol.

IFG is an azasugar that binds both wild-type and mutant forms of GCase, resulting in stabilization and increased cellular and lysosomal levels,49,50 particularly for the N370S variant .50,51 Similarly, the cellular levels of a number of other missense mutant forms of GCase, including L444P, are also increased in response to incubation with IFG.50,52,53 Preclinical in vivo studies evaluated the effects of IFG in different transgenic mouse models of Gaucher disease homozygous for the mutations L444P, V394L, N370S, D409H, or D409V.50,53,54 Overall, IFG administration (at different doses) led to statistically significant increases in GCase activity in

10 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW liver, spleen, lung, bone, and brain, as well as in liver macrophages. In the L444P and N370S models, IFG administration resulted in reduced liver and spleen weights, providing evidence of in vivo efficacy. In the V394L model, which to some extent mimics neuronopathic forms of the disease, increases in GCase protein levels and activity in brain, reduced neuroinflammation, delayed onset of neurological disease, and increased life spans were demonstrated.50

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Ambroxol, an approved expectorant, was identified from a screen of 1,040 FDA-approved drugs

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by its ability to stabilize wild-type GCase against thermal denaturation.55 Ambroxol also showed

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pH-dependent affinity for GCase, with decreasing inhibition at lysosomal pH values. In in vitro

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studies, Ambroxol elevated the cellular and lysosomal levels of multiple GCase mutants in patient-derived cells expressing R120W, R131C, N188S, G193W, F213I, N370S, L444P, and/or

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P415R.56,57 In vivo, in N370S or L444P transgenic mice, daily subcutaneous injections of

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Ambroxol for 14 days resulted in consistently elevated GCase levels in spleen and liver.54

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Pompe disease (acid maltase deficiency, glycogen storage disease type II) is an LSD caused by mutations in the GAA gene that encodes acid α-glucosidase (GAA; EC 3.2.1.20),58 an enzyme involved in lysosomal glycogen catalysis. Deficiency in GAA activity results in generalized glycogen accumulation in heart, skeletal muscle, and other tissues.58 Currently, ERT with recombinant human GAA (rhGAA; alglucosidase alfa), is the only approved treatment for Pompe disease. However, ERT shows limitations, particularly in terms of restricted bioavailability, insufficient correction of disease pathology in some muscles, tolerability issues, and immunogenicity.

11 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Small molecule chaperones have been proposed as a potential alternative to ERT for the treatment of Pompe disease. Some of the approximately 150 mutant forms of GAA are responsive to 1-deoxynojirimycin (DNJ, AT2220, duvoglustat) and N-butyldeoxynojirimycin (NB-DNJ, miglustat).59,60,61 Mechanistically, DNJ has multiple modes of action during the synthesis and maturation of mutant GAA, including increased specific activity prior to proteolytic processing in lysosomes, facilitated export from the ER with subsequent trafficking and processing through the secretory pathway to lysosomes, and stabilization of mature isoforms

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in lysosomes.62 The in vivo effects of DNJ were tested in a mouse model of Pompe disease that

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expresses the human mutant GAA transgene P545L on a Gaa KO background.62 Daily oral administration of DNJ to the transgenic mice resulted in significant and dose-dependent

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increases in GAA activity with concomitant reduction in tissue glycogen levels.

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Pyrimethamine, an approved antimalarial drug, was identified as a potential chaperone of βhexosaminidase (β-Hex; EC 3.2.1.52), a lysosomal hydrolase that cleaves N-acetylgalactosamine

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from glycosphingolipids, various oligosaccharides, and glycoproteins.63 β-Hex deficiency results in two progressive neurodegenerative GM2 gangliosidoses (Tay-Sachs disease and Sandhoff disease), for which there are currently no effective treatment options.

In another severe neurodegenerative LSD that results from deficiency of β-galactosidase (EC 3.2.1.23), GM1 gangliosidosis, the small molecules N-octyl-4-epi-β-valienamine (NOEV) and 5N,6S-(N'-butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin were shown to significantly enhance β-galactosidase activity both in vitro and in transgenic animal models of 12 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW the disease.64,65 Interestingly, NOEV administration starting at the early stage of disease resulted in a reduced rate of disease progression, arrest of neurological involvement, and prolonged survival of treated animals. The Other Side of the Coin: Limitations of PCT Although PCT has several potential advantages over existing therapies, and has already shown clinical efficacy in one LSD (i.e., Fabry disease), there are some challenges to be addressed by

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future research.

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In most cases chaperones are reversible competitive inhibitors of target enzymes. To date, most

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pharmacological chaperones that have been identified bind to the active site of their target

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enzyme, thus acting as inhibitors. Initially, the idea of using small molecule, active-site inhibitors to increase total cellular enzyme activity was counterintuitive. However, in many cases, if used

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appropriately, net gains in in situ lysosomal activity and reduced substrate levels can be

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realized.12,13 In fact, some pharmacological chaperones bind with high affinity to their target enzyme at neutral pH, but show lower binding affinity at acidic pH, thus favoring dissociation

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and substrate turnover in the lysosome. In addition, once delivered to the lysosome, high concentrations of accumulated substrate can compete with the chaperone for binding to the target enzyme, thus facilitating substrate turnover rather than enzyme inhibition.

Increases in residual activity may be too low for significant benefit. Preclinical studies have shown variable increases in activity in the presence of chaperones. While some enzyme variants appear to respond quite well, others show only minor net increases in activity (i.e., enhancement), lysosomal trafficking, and enzyme processing/maturation. For some diseases and for some specific mutations, the enhancement in residual activity obtained with PCT may not be 13 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW sufficient to translate into significant benefit for patients, particularly if tissue pathology is already fully established. However, insufficient correction of the enzymatic defect and pathology in specific tissues is also a challenge with ERT and other approaches (see above).

Only a fraction of mutations are responsive to chaperone therapy. Not all LSD patients have missense mutations and not all missense mutations are responsive to

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chaperones. The determinants of chaperone response have been analyzed and depend both on the

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type of mutation and on the specific chaperone molecule tested.66,67,68,69 In silico characterization

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of the molecular interactions between enzymes and chaperones may allow prediction of

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responsiveness of mutant enzymes to chaperones.66,67 It is reasonable to expect that mutations

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that lead to major reductions in physical stability, that prevent folding, or that affect catalytic

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activity may not be responsive to active-site pharmacological chaperones. Molecules that interact

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with different domains of the protein (including non-catalytic domains) may have the potential to expand the spectrum of responsive mutations.70

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The rate of responsive mutations varies in the different LSDs. For Fabry disease, in which most mutations are private, with none showing high prevalence,71 the fraction of missense mutations that are potentially responsive is estimated to be 30-50%. For Gaucher disease, more than 70% of patients within the Ashkenazi Jewish population carry at least one N370S allele, while 38% of non-Jewish patients carry the L444P allele.72,73 Both mutations are, in principle, responsive to PCT. For Pompe disease, it is possible that 10-15% of patients would be amenable to PCT.61 However, to estimate the true percent of LSD patients that are amenable to PCT, it should be

14 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW considered that only a fraction of mutations cause premature termination of translation, gene deletions, rearrangements, splicing mutations, or are unknown. Interestingly, in some cases endogenous wild-type enzymes also show a response to chaperones, while in other instances they do not. The reasons for this are not clear, with several factors possibly playing roles, including the overall folding efficiency for the enzyme/protein of interest, which may be different across cell types and species. For example, DNJ enhances wild-type Gaa in mice and rats very robustly, but less so in monkeys; virtually no enhancement of wild-type

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GAA was seen in WBCs of healthy human volunteers (unpublished results). As mentioned

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above, how well the chaperone interacts and coordinates key amino acid side chains to confer enhanced stability may also play a role. The molecular interactions that lead to the desirable

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chaperone properties of DGJ for α-Gal A have been described, and are different than the

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interactions between α-Gal A and galactose, a low affinity ligand with generally poorer

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chaperone properties.67

How to circumvent the limitations of PCT?

chaperones.

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Different strategies have been devised to address the potential limitations of pharmacological

Discontinuous administration of chaperones. The lysosomal half-life of the target enzyme (typically days) relative to that of the pharmacological chaperone (typically hours) can be used to develop optimal dose and administration regimens that allow maximal substrate turnover, thereby addressing concerns on potential inhibition. For instance, in hR301Q α-Gal A Tg/KO mice, discontinuous oral administration of the chaperone DGJ (cycles of 4 days with DGJ followed by 3 days without DGJ to allow for tissue clearance of the chaperone), resulted in greater substrate reduction compared to daily administration. The shorter tissue half-life of DGJ 15 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW compared to that of α-Gal A could be exploited to stabilize α-Gal A in the ER and promote lysosomal trafficking during DGJ administration, and to maximize lysosomal α-Gal A activity and substrate turnover during periods in the absence of DGJ, thus producing a larger net gain in lysosomal enzyme activity.46

Pharmacological chaperones that target allosteric sites. Current research is focused on the

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identification of pharmacological chaperones that bind allosteric sites of target enzymes. The

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stabilization mechanism of these allosteric compounds would be, in principle, similar to the

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active site-directed compounds, and require a sufficient binding affinity to increase the stability

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of the mutant enzyme structure. However, allosteric chaperones would have the additional

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advantage that they can remain bound to the enzyme during catalysis without risk of inhibition.

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Examples of allosteric pharmacological chaperones are now being reported.

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α-Gal A contains an allosteric site that selectively binds the β-anomer of D-galactose.74 For GCase, compounds that enhance enzymatic activity, possibly by binding to sites other than the catalytic site, also have been described.75 An in silico study identified regions on the surface of GCase that may host allosteric ligands.76 Allosteric pharmacological chaperones also have been documented for GAA. A high-throughput screen of more than 200,000 compounds identified 1(3,4-dimethoxybenzyl)-6-propyl-2-thioxo-2,3,5,6,7,8-hexahydropyrimido[4,5-d]pyrimidin4(1H)-one (ML247).70. In addition, Porto et al showed that N-acetyl-cysteine, and the related compounds N-acetyl-serine and N-acetyl-glycine, increased the physical stability of the enzyme

16 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW as a function of pH and temperature, and enhanced the activity of the enzyme in cultured Pompe fibroblasts.70 Combination therapies. Although PCT has been designed to rescue endogenous mutant misfolded proteins, studies suggest that chaperones are able to increase the stability of the wildtype enzymes that are commonly used for ERT. This effect has the potential to translate into enhanced efficacy of ERT and to address some of the limitations of chaperones. To this end, preclinical studies demonstrated that pharmacological chaperones enhance enzyme stability,

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lysosomal trafficking, and/or processing in Pompe, Fabry, and Gaucher cultured cells incubated

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with the respective recombinant enzymes, as well as in Pompe and Fabry animal

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models.70,78,79,80,81

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In cultured fibroblasts from Pompe patients, co-incubation of the chaperone NB-DNJ with

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rhGAA ERT resulted in greater correction of enzyme activity and increased amounts of the recombinant enzyme in lysosomal compartments.82 Co-administration of DNJ with rhGAA ERT

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to Gaa KO mice resulted in significantly greater rhGAA levels in plasma, and greater enzyme uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone, indicating enhanced efficacy.82

Similarly, co-incubation with DGJ significantly increased the physical stability of α -Gal A ERT at neutral pH, and prevented loss of activity in human whole blood ex vivo relative to incubation without DGJ.81 Furthermore, co-incubation of Fabry patient-derived fibroblasts with DGJ and α -Gal A ERT resulted in greater cellular enzyme levels, and 17 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW greater GL-3 reduction, compared to incubation with α -Gal A alone.59,80,81,82,83 In vivo, co-administration of DGJ and α -Gal A ERT to rats and Gla KO mice resulted in significantly greater plasma exposure of active enzyme, higher tissue levels of active enzyme, and greater reduction of tissue GL-3, when compared to administration of α -Gal A alone.80 Similar effects were seen when DGJ was co-formulated with Fabry ERT and infused into rats and Gla KO mice, offering a potential alternative route of administration

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for the chaperone.84

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The synergy between chaperones and ERT may help broaden the use of PCT beyond

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the rescue of mutant enzymes. With the combination of PCT and ERT, the effect of chaperones is directed towards the ERT, and is mutation-independent. Thus, in

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principle, this approach may be beneficial to any patient on ERT, if treated with the

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specific chaperone. In addition, the administration of the chaperone could be restricted only to the time of ERT infusions, thus reducing the risk of any potentially undesired

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events that may be related to the chronic use of the chaperone.

Translation of PCT into Clinical Studies Monotherapy. After preclinical studies, pharmacological chaperone research is in a phase of clinical translation, with five chaperones for four LSDs now evaluated in clinical studies, and others showing promise (TABLE 1).

18 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Galactose was the first pharmacological chaperone investigated for Fabry disease. Every-otherday intravenous infusion of 1 g/kg galactose improved cardiac function in a male patient who presented with severe cardiomyopathy.85 After two years, cardiac transplantation was unnecessary, offering the first proof-of-concept for PCT in an LSD. DGJ, the active component of the investigational drug migalastat hydrochloride (Amicus Therapeutics, Cranbury, NJ) is being developed as a treatment for Fabry disease. After successfully completing Phase 1 studies,86 two Phase 2 studies enrolled 9 male Fabry subjects to

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investigate the safety and efficacy of 150 mg migalastat every other day (QOD).87,88,89 Increased

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α-Gal A activity and decreased GL-3 were seen in white blood cells (WBCs), skin, urine, and/or kidney in the 6 subjects that expressed mutant forms of α-Gal A that were responsive to

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migalastat in an in vitro cell-based assay (i.e., amenable); the other 3 subjects had non-amenable

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mutant forms.89,90 In another Phase 2 study conducted in females,91,92 subjects with migalastat-

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amenable mutant forms showed greater responses in vivo based on reduced urine GL-3 and reduced renal peritubular capillary inclusions. Across all Phase 2 studies, migalastat was

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generally safe and well tolerated.93,94

As such, Phase 3 studies were initiated to further evaluate the effect of 150 mg migalastat QOD. To date, data presented at scientific conferences have shown the efficacy of migalastat in stabilizing renal function, reducing left ventricular mass, and improving gastrointestinal symptoms in Fabry disease patients with amenable mutations. Long-term open-label extension studies of migalastat are ongoing.95,96,97,98,99,100

19 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW As discussed, two pharmacological chaperones have advanced to the clinic as potential therapies for Gaucher disease: IFG and Ambroxol. In Phase 1 studies, IFG showed dose-related elevations of up to 3.5-fold in WBC GCase levels. Subsequently, two Phase 2 studies were initiated. In a 4week study conducted in subjects with twelve different GBA1 mutations (including N370S and L444P) previously treated with ERT,101 WBC GCase activity increased in 20 of 26 subjects. As expected for a short-term study, disease markers including platelet, hemoglobin, glucosylceramide, and chitotriosidase levels were unchanged. In another 6-month Phase 2 study

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in patients who had never received ERT,102 all subjects showed an increase in WBC GCase

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levels, but only one attained improvements in clinical measures.

The tolerability and efficacy of once-daily 150 mg Ambroxol in 12 Gaucher patients were

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evaluated in an investigator-sponsored pilot study.103 These subjects were ERT-naive, with 11

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being N370S homozygotes. Of the 9 subjects that completed the 6-month study, none showed deterioration of Gaucher-related parameters (e.g., weight, hemoglobin, platelet count,

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liver/spleen volume, and chitotriosidase activity), and three continued therapy for an additional 6 months. These three also showed mean reductions of ~15% and ~40% in spleen volume and chitotriosidase activity, respectively. In one subject, platelet counts increased over 50%.

Pharmacological chaperones also have been proposed as a treatment for Pompe disease.59,60,61,62 To this end, Phase 1 studies with DNJ showed that the drug was generally safe and well tolerated. A Phase 2 study104 conducted in adult Pompe subjects enrolled into 1 of 3 dose cohorts (2.5 to 5 grams, with discontinuous administration protocols) was terminated due to severe

20 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW adverse events in 2 subjects (muscle weakness) that resolved following DNJ withdrawal. These adverse events were deemed due to DNJ-mediated inhibition of endogenous GAA activity. A follow-up Phase 1 study was conducted to evaluate the muscle pharmacokinetics of DNJ after a single 1000 mg oral dose.96 DNJ rapidly appeared and remained in muscle for over one week at concentrations that were in excess of its IC50 value for inhibition of GAA. The muscle profile of DNJ suggests that the doses selected for the Phase 2 study were too high, and that an appropriate

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balance between chaperoning and inhibition was not achieved.

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The potential benefits of pyrimethamine on endogenous levels of mutant β-Hex were

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investigated in a 16-week Phase 1/2 study in late-onset GM2 gangliosidosis patients.105 Eight of

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11 subjects completed the study, and showed up to 4-fold increases in β-Hex activity in WBCs at

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doses ≤50 mg per day. Increased ataxia, lack of coordination, and seizures were observed in most

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subjects at higher doses. These data indicate that pyrimethamine can enhance β-Hex activity in peripheral cells of late-onset GM2 gangliosidosis patients at doses lower than those associated

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with adverse effects.

Combination Therapy (ERT + chaperones). Given the promising preclinical results, the potential of therapeutic protocols based on the combination of ERT and chaperones also has been evaluated in clinical studies. The first published clinical study on the combination of rhGAA ERT and the chaperone NB-DNJ (miglustat) in Pompe disease reported on the results of a collaborative trial in 13 patients with different presentations (3 infantile-onset, 10 late-onset).106 All patients had been previously treated with ERT for variable periods (1-8 years). The primary 21 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW endpoint of the study was to obtain higher levels of blood GAA activity with the combination of rhGAA and the chaperone, compared to the activities obtained with ERT alone. GAA activity was measured by tandem-mass spectrometry in dried blood spots. In 11 patients, the combination treatment resulted in GAA activities greater than 1.85-fold the activities seen with ERT alone. In the whole patient population, GAA activity was significantly increased at 12 hours, 24 hours, and 36 hours. In another Phase 2 study conducted by Amicus Therapeutics, Pompe subjects were orally administered a single dose of 50, 100, 250, or 600 mg DNJ 1 hour prior to ERT infusion.

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Dose-dependent increases in plasma GAA activity were observed for all subjects, attaining 1.5-

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to 2.8-fold greater exposures compared to ERT alone. In muscle biopsy samples taken 3 or 7 days after administration, increases in total GAA activity were observed in 16 of 24 subjects with

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evaluable data.107 These results indicate consistency with preclinical data obtained in rats and

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Gaa KO mice that showed a longer circulating half-life of GAA when co-administered with

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chaperone.82

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Lastly, a Phase 2 study108 was initiated by Amicus Therapeutics to investigate the effects of 150 and 450 mg migalastat HCl when administered 2 hours prior to infusion of α-Gal A ERT.109 As seen in preclinical studies, plasma exposures of active α-Gal A were 1.2- to 5.0-fold greater in 22 of the 23 male Fabry subjects following co-administration compared to levels seen following administration of ERT alone. Migalastat also led to greater total α-Gal A activity in the skin of 19 of the 23 subjects relative to ERT alone, as measured in biopsies collected 24 hours postinfusion.

22 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Conclusions The complexity of LSD pathophysiology has made the development of therapies a major challenge. Presently, none of the therapeutic approaches that are already approved for clinical use have proven suitable to treat all LSDs, or all patients with a specific disorder. PCT appears to have the potential to address some of the medical needs posed by LSDs and the limitations of currently available therapies. Although for some LSDs PCT has gone through remarkable progress and has shown efficacy and safety in one LSD, Fabry disease, further development and

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innovation is expected. Future research will be directed towards the identification of novel drugs

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with different chaperoning profiles to target a larger number of mutations. In addition, it will be important to identify new allosteric chaperones to exploit the full potential of chaperones and

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obtain the greatest clinical efficacy across LSDs. It is reasonable to think that therapeutic

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protocols will need to be tailored for individual patients with LSDs, possibly after in silico or in

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vitro evaluations, in order to predict the response of individual patients, and that protocols based on the association of different therapeutic agents may be more efficacious and result in

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synergistic effects.

Acknowledgements

Kenneth J. Valenzano is employed by Amicus Therapeutics and is shareholder in the company. The support of the Telethon Foundation (grant TGPMT4TELD to G.P.), and Programma Operativo Nazionale (PON) 01_00862 to GP, is gratefully acknowledged.

23 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

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ACCEPTED ARTICLE PREVIEW 55. Maegawa GH, Tropak MB, Buttner JD, Rigat BA, Fuller M, Pandit D et al. (2009). Identification and characterization of ambroxol as an enzyme enhancement agent for Gaucher disease. J Biol Chem 284: 23502-23516. 56. Luan Z, Li L, Higaki K, Nanba E, Suzuki Y, Ohno K (2013). The chaperone activity and toxicity of ambroxol on Gaucher cells and normal mice. Brain Dev 35: 317-322. 57. Bendikov-Bar I, Maor G, Filocamo M, Horowitz M (2013). Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase. Blood Cells Mol Dis 50: 141-145.

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30 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

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ACCEPTED ARTICLE PREVIEW 71. Desnick RJ, Ioannou YA, Eng CM (2001). α-Galactosidase A deficiency: Fabry disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Disease. 7th edn. McGraw-Hill: New York, pp 3733-3774. 72. Grabowski GA (1997). Gaucher disease: gene frequencies and genotype/phenotype correlations. Genet Test 1: 5-12. 73. Horowitz M, Pasmanik-Chor M, Borochowitz Z, Falik-Zaccai T, Heldmann K, Carmi R et al. (1998). Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish

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ACCEPTED ARTICLE PREVIEW 79. Porto C, Cardone M, Fontana F, Rossi B, Tuzzi MR, Tarallo A et al. (2009). The pharmacological chaperone N-butyldeoxynojirimycin enhances enzyme replacement therapy in Pompe disease fibroblasts. Mol Ther 17: 964-971. 80. Porto C, Pisani A, Rosa M, Acampora E, Avolio V, Tuzzi MR et al. (2012). Synergy between the pharmacological chaperone 1-deoxygalactonojirimycin and the human recombinant alpha-galactosidase A in cultured fibroblasts from patients with Fabry disease. J Inherit Metab Dis 35: 513-520.

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81. Benjamin ER, Khanna R, Schilling A, Flanagan JJ, Pellegrino LJ, Brignol N et al. (2012).

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Co-administration with the pharmacological chaperone AT1001 increases recombinant human αgalactosidase A tissue uptake and improves substrate reduction in Fabry mice. Mol Ther 20: 717-

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726.

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82. Khanna R, Flanagan JJ, Feng J, Soska R, Frascella M, Pellegrino LJ et al. (2012). The

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pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake

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and glycogen reduction in a mouse model of Pompe disease. PLoS One 7: e40776

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83. Pisani A, Porto C, Andria G, Parenti G (2014). Synergy between the pharmacological chaperone 1-deoxygalactonojirimycin and agalsidase alpha in cultured fibroblasts from patients with Fabry disease. J Inherit Metab Dis 37: 145-146. 84. Lun Y, Xu S, Brignol N, Chang K, Frascella M, Garcia A et al. (2014). Next-generation enzyme replacement therapy for Fabry disease: co-formulation of migalastat HCl with a proprietary recombinant human α-galactosidase A leads to enhanced enzyme uptake and GL-3 reduction in Fabry mice. J Inherit Metab Dis 37: S152 (abstract P-148).

33 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW 85. Frustaci A, Chimenti C, Ricci R, Natale L, Russo MA, Pieroni M et al. (2001). Improvement in cardiac function in the cardiac variant of Fabry's disease with galactose-infusion therapy. N Engl J Med 345: 25-32. 86. Johnson FK, Mudd PN Jr, Bragat A, Adera M, Boudes P (2013) Pharmacokinetics and Safety of Migalastat HCl and Effects on Agalsidase Activity in Healthy Volunteers. Clin Pharmacol Drug Dev 2: 120–132. 87. ClinicalTrials.gov ID NCT00283959

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89. Germain DP, Giugliani R, Hughes DA, Mehta A, Nicholls K, Barisoni L et al. (2012). Safety and pharmacodynamic effects of a pharmacological chaperone on α-galactosidase A activity and

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globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies.

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90. Wu X, Katz E, Della Valle MC, Mascioli K, Flanagan JJ, Castelli JP et al. (2011). A

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pharmacogenetic approach to identify mutant forms of α-galactosidase A that respond to a

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pharmacological chaperone for Fabry disease. Hum Mutat 32: 965-977. 91. ClinicalTrials.gov ID NCT003045120 92. Giugliani R, Waldek S, Germain DP, Nicholls K, Bichet DG, Simosky JK et al. (2013). A Phase 2 study of migalastat hydrochloride in females with Fabry disease: selection of population, safety and pharmacodynamic effects. Mol Genet Metab 109: 86-92. 93. ClinicalTrials.gov ID NCT00526071 94. Hughes D, Adera M, Castelli J, Bragat A, Marsden DL, Boudes PB (2010) Preliminary Long-Term Safety, Tolerability, and Assessments of Renal Function of Adult Fabry Patients

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ACCEPTED ARTICLE PREVIEW Receiving Treatment with AT1001 (Migalastat Hydrochloride), a Pharmacological Chaperone, for Up to 3 Years. J Inherit Metab Dis 33 (Suppl 1): S148 (abstract 473-O). 95. ClinicalTrials.gov: NCT00925301 96. Adera M, Overton C, Boudes P (2011) A double-blind, randomized, placebo-controlled study to evaluate the efficacy, safety and pharmacodynamics of AT1001 in patients with Fabry disease and AT1001-responsive GLA mutations. Mol Genet Metab 102(2):S4-S5 (abstract 4). 97. Barisoni L, Jennette JC, Colvin R, Sitaraman S, Bragat A, Castelli J et al. (2012). Novel

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quantitative method to evaluate globotriaosylceramide inclusions in renal peritubular capillaries

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by virtual microscopy in patients with fabry disease. Arch Pathol Lab Med 136: 816-824. 98. Nicholls K, Germain DP, Feliciani C, Shankar S, Ezgu F, Janmohamed SG et al. (2013).

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Phase 3 study of migalastat HCl for Fabry disease: Stage 1 results. Mol Genet Metab 108(2): S70

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(abstract 168).

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99. Barlow C, Castelli J, Benjamin ER, Yu J, France N, Ludington E et al. (2014). Phase 3

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FACETS study of migalastat HCl for Fabry disease: post hoc GLA mutation-based identification

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of subjects likely to show a drug effect. Mol Genet Metab 111(2): S24 (abstract 24). 100. Barlow C (2014) Clinical results using a GLP-validated pharmacogenetic test identifies subjects responsive to migalastat HCl in the FACETS study. Mol Genet Metab 111(2): S23 (abstract 23). 101. ClinicalTrials.gov ID NCT01218659 102. ClinicalTrials.gov ID NCT00433147 103. ClinicalTrials.gov ID NCT00446550 104. Zimran A, Altarescu G, Elstein D (2013). Pilot study using ambroxol as a pharmacological chaperone in type 1 Gaucher disease. Blood Cells Mol Dis 50: 134-137. 35 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW 105. ClinicalTrials.gov ID NCT00688597 106. Clarke JT, Mahuran DJ, Sathe S, Kolodny EH, Rigat BA, Raiman JA et al. (2011). An open-label Phase I/II clinical trial of pyrimethamine for the treatment of patients affected with chronic GM2 gangliosidosis (Tay-Sachs or Sandhoff variants). Mol Genet Metab 102: 6-12. 107. Parenti G, Fecarotta S, la Marca G, Rossi B, Ascione S, Donati MA et al. (2014). A chaperone enhances blood α-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy. Mol Ther 22: 2004-2012.

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108. Kishnani P, Tarnopolsky M, Sivakumar K, Roberts M, Byrne B, Goker-Alpan O et al.

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(2013). A phase 2a study to investigate drug–drug interactions between escalating doses of

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109. ClinicalTrials.gov ID NCT01196871

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AT2220 (duvoglustat hydrochloride) and acid alfa-glucosidase in subjects with Pompe disease.

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110. Warnock D, Bichet D, Holida M, Goker-Alpan O, Nicholls K, Thomas M et al. (2013). A

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phase 2A study to investigate the effect of a single dose of migalastat HCl, a pharmacological

(abstract 248).

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chaperone, on agalsidase activity in subjects with Fabry disease. Mol Genet Metab 108(2): S96

111. Tropak MB, Reid SP, Guiral M, Withers SG, Mahuran D (2004). Pharmacological enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff Patients. J Biol Chem 279: 13478-13487. 112. Tropak MB, Mahuran D. (2007). Lending a helping hand, screening chemical libraries for compounds that enhance beta-hexosaminidase A activity in GM2 gangliosidosis cells. FEBS J 274: 4951-4961.

36 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW 113. Knight EM, Williams HN, Stevens AC, Kim SH, Kottwitz JC, Morant AD et al. (2015). Evidence that small molecule enhancement of β-hexosaminidase activity corrects the behavioral phenotype in Dutch APP(E693Q) mice through reduction of ganglioside-bound Aβ. Mol Psychiatry.20: 109-117. 114. Feldhammer M, Durand S, Pshezhetsky AV. (2009). Protein misfolding as an underlying molecular defect in mucopolysaccharidosis III type C. PLoS One 13: e7434. doi: 10.1371 115. Dawson G, Schroeder C, Dawson PE (2010). Palmitoyl:protein thioesterase (PPT1)

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inhibitors can act as pharmacological chaperones in infantile Batten disease. Biochem Biophys

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Res Commun.395: 66-69.

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37 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

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TABLE 1. Current stage of development of select pharmacological chaperones Therapeutic Agent (route of administration, as applicable) Galactose (intravenous)

Development Status / Clinical Trial Study ID#

ip t

Orphan Drug Designation

MONOTHERAPY

N/A

cr

Disease

Refs

Università Cattolica del Sacro Cuore, Roma, Italy

85

Amicus Therapeutics

89, 92

Amicus Therapeutics

109

us

Pilot clinical study

Company, Sponsor

an

MONOTHERAPY

d

m

Phase 2: NCT00214500 (FAB-CL-201) NCT00283959 (FAB-CL-202) NCT00283933 (FAB-CL-203) NCT003045120 (FAB-CL-204) NCT00526071 (FAB-CL-205)

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US: 02/25/2004 EU: 05/22/2006

A

cc

Migalastat HCl / AT1001 / 1-deoxygalactonojirimycin / DGJ (oral)

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Fabry

Phase 3: NCT00925301 (AT1001-011) NCT01218659 (AT1001-012) NCT01458119 (AT1001-041) NCT02194985 (AT1001-042) COMBINATION THERAPY (Phase 2) + intravenous agalsidase alfa or beta: NCT01196871 (AT1001-013)

38 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Table 1 (continued) MONOTHERAPY (Phase 2) US: 01/10/2006 EU: 10/23/2007

NCT00875160 (GAU-CL-104) NCT00433147 (GAU-CL-201) NCT00446550 (GAU-CL-202) NCT00813865 (GAU-CL-202X)

Ambroxol (oral)

US: 06/29/2011 EU: N/A

MONOTHERAPY (Phase 1/2)

MONOTHERAPY (Phase 2)

an

NCT00688597 (POM-CL-201)

103

Amicus Therapeutics Amicus Therapeutics

107

Università degli Studi di Napoli Federico II, Italy

106

d

+ intravenous alglucosidase alfa: NCT01380743 (AT2220- 010)

NOEV (oral)

COMBINATION THERAPY (Phase 2) + intravenous alglucosidase alfa: EudraCT n. 2010-024647-32

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N/A

A

Miglustat (oral)

ep

te

Pompe

GM1 gangliosidosis

ExSAR Corp.

COMBINATION THERAPY (Phase 2)

m

Duvoglustat HCl / AT2220 / US: 06/18/2007 1-deoxynojirimycin / DNJ EU: N/A (oral)

Amicus Therapeutics

cr

NCT01463215

us

Gauc her, type I

ip t

Afegostat tartrate / AT2101 / isofagomine / IFG (oral)

COMBINATION THERAPY (Phase 1/2) + intravenous alglucosidase alfa: NCT02185651

N/A

MONOTHERAPY Preclinical cell-based and in vivo studies

University of Florida / Amicus Therapeutics

N/A

64

39 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW Table 1 (continued) MONOTHERAPY

N-acetylglucosamine thiazoline (NGT)

N/A

AdNDJ / OT1001 (oral)

N/A

Pyrimethamine (oral)

US: 08/16/2011 EU: N/A

MPS IIIC

Glucosamine

N/A

Batten

CS38

N/A

MONOTHERAPY

ip t

Preclinical cell-based and in vivo studies MONOTHERAPY (Phase 1/2)

us

cr

NCT01102686

MONOTHERAPY

Preclinical cell-based studies only

110, 111

N/A

110, 112

The Hospital for Sick Children, Canada

105

N/A

113

N/A

114

an

GM2 gangliosidosis

Preclinical cell-based studies only

N/A

m

MONOTHERAPY

te

d

Preclinical cell-based studies only

A

cc

ep

N/A, not applicable; US, United States; EU, European Union

40 © 2015 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

Figure legends FIGURE 1 The cellular pathways that control folding of lysosomal enzymes. During synthesis, proteins (in this case, lysosomal enzymes or proteins) are co-translationally assisted by molecular chaperones and folding factors (e.g., heat-shock proteins) that interact with partially folded, aggregation-prone structural motifs of the nascent protein. Upon recognition and binding to the nascent polypeptide, molecular chaperones can stabilize protein conformation, inhibit premature misfolding, and prevent aggregation. Enzymes that

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are correctly folded and stable pass the quality control (QC) of the ER, exit the ER efficiently, and

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traffic to lysosomes. Mutant, misfolded enzymes may undergo ER retention, or be recognized by the ER QC, retro-translocated to the cytosol, and degraded by ER-associated degradation systems (ERAD).

an

FIGURE 2

m

Mechanism of action of pharmacological chaperones. Pharmacological chaperones (hexagons) are small

d

molecule ligands that selectively bind and stabilize otherwise unstable enzymes and enhance or partially

ep te

restore their folding and stability. Enzymes that are rescued by pharmacological chaperones can be

FIGURE 3

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normally trafficked, thus increasing residual activity in lysosomes.

Correlations between residual activity and LSD phenotype. For most LSDs, correlations have been observed between residual enzyme activity and disease severity. It has been speculated that substrate storage occurs if residual activity falls below a certain threshold. For several LSDs, it has been assumed that a threshold activity of approximately 10% is sufficient to prevent storage, while 3% to 5% residual activity is associated with attenuated phenotypes.

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ACCEPTED ARTICLE PREVIEW

Figure 1

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ACCEPTED ARTICLE PREVIEW

Figure 2

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ACCEPTED ARTICLE PREVIEW

Figure 3

© 2015 The American Society of Gene & Cell Therapy. All rights reserved