Treatment of Lysosomal Storage Diseases: Recent Patents and Future Strategies

Send Orders for Reprints to [email protected] Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, 8, 9-25 9 Treatment of Lysosomal Storage Diseases: Recent Patents and Future Strategies

1,* 1 1 1 Saida Ortolano , Irene Viéitez , Carmen Navarro and Carlos Spuch

1 Neuroscience Research Group, Instituto de Investigación Biomedica de Vigo (IBIV), Xerencia de Xestión Integrada de Vigo - SERGAS, Vigo, Spain

Received: October 31, 2013; Accepted: January 6, 2014; Revised: January 10, 2014 Abstract: Lysosomal storage diseases (LSDs) are a group of rare genetic multisystemic disorders, resulting in deficient lysosomal activity. These pathologies are characterized by progressive accumulation of storage material within the lysosomes, ultimately leading to organ dysfunctions. LSDs patient’s clinical outcomes have significantly improved, since the advent of enzyme replacement therapy (ERT). ERT is approved worldwide for 6 LSDs: Gaucher disease, Fabry disease, Mucopolysaccharidosis types I, II, and VI, and Pompe disease. The efficacy and safety of ERT for LSDs has been confirmed by extensive clinical trials, however therapy with infused protein is life-long and disease progression is still observed in treated patients. Obstacles to successful ERT, such as immune reactions against the infused enzyme, miss-targeting of recombinant enzymes, and difficult delivery to crucial tissues (i.e. brain and bone), determine the need for further research, in order to ameliorate therapeutic strategies. Viral gene therapy, stem cell based therapy, pharmacological chaperones and could be considered essential tools for future improvement of recombinant enzyme trafficking and targeting. This review will discuss recent patents and new strategic approaches for enzyme delivery to highlight the most relevant aspects, concerning next generation LSDs treatment. Keywords: Enzyme replacement therapy, Fabry disease, Gaucher disease, gene therapy, hematopoietic stem cells transplantation, lysosomal storage diseases, pharmacological chaperons, substrate reduction therapy.

INTRODUCTION severity. For all these reasons, while the variety of therapeutic options for patients has greatly grown, the effective clini- Lysosomal Storage Diseases (LSDs) are inherited meta- cal management of these disorders remains an unfulfilled bolic multisystemic conditions, affecting lysosomal function. challenge. LSDs comprise a group of more than 50 rare disorders The multisystemic nature of these pathologies determines which are caused by specific mutations in genes encoding that healthcare professionals of different expertise (such as lysosomal enzymes, responsible for the degradation of a nephrologists, cardiologists, neurologists, pediatricians, wide variety of glycolipids, oligosaccharides, proteins and metabolic physicians, dermatologists, internists and general glycoproteins (Table 1). Although often catalytically compe- practitioners) are involved in medical assistance to LSDs tent, the mutated enzymes are unable to overcome the effi- patients during their lifetime. In order to provide the best cient quality control of the endoplasmic reticulum, ultimately possible management to affected patients, physicians should causing reduced lysosomal trafficking, substrate accumula- be well-aware of the signs and symptoms of individual tion and cellular dysfunction. Storage accumulation in dif- LSDs, as well as of their possible evolution and the time of ferent tissues is thought to underlie the clinical manifesta- onset. However, patients’ identification is extremely difficult tions of LSDs and to eventually be the cause of organ failure during routine consultations, due to the low incidence of [1]. As a group, many of LSDs are progressive in nature, LSDs in the population. Therefore, while collectively the share similar clinical evidences and in many cases leads to overall incidence of LSDs is relatively frequent, occurring in premature death. Common signs seen in LSDs include skele- about 1 in 4000 -8000 live births individually the LSDs are tal defects, organomegaly, central nervous system defects rare with incidences ranging from 1:57000 (Gaucher disease) and coarsening of hair and facial features. Nevertheless, each to 1:4,200000 (sialidosis) [2]. disease also has unique characteristics marked by its biochemistry and pathophysiology. Phenotypic heterogeneity is Even though the incidence of LSDs is probably underes- also evident in individuals who share the same disease, due timated, as shown in recent screening studies enrolling new- to multiple organs involvement and differences in symptoms borns [3], delayed diagnosis is a commonplace in their LSDs, due to the non-specific nature of disease manifesta- *Address correspondence to this author at the Neuroscience Research Group tions arising during childhood. To provide patient an early NC-CHUVI, Instituto de Investigación Biomedica de Vigo (IBIV), Bio- diagnosis, which is strictly related to efficacy of the available medical Research Unit, Hospital Rebullón (CHUVI), Puxeiros s/n, 36415 treatments, is hence another partially unmet goal for clini- MOS (Pontevedra), Spain; Tel: +34 986824871; Fax: +34 986487516; cians. E-mail: [email protected]

Table 1. Molecular Basis of Frequent LSDs.

DISEASE GENE ENZYME STORAGE PRODUCT

Tay-Sachs HEX A -Hexosaminidase A GM2, Condroitin sulfate

Sandhoff HEX B -Hexosaminidase A/B GL4, GA2, GM2

Fabry GLA -Galactosidase A Gb3 LysoGb3

Gaucher GBA -Glucosidase Glucocerebrosidase

GM1 Gangliosidosis GLB1 -Galactosidase GM1

Krabbe GALC Galactosylcerebrosidase GalCer, Psychosine

Pompe GAA -Glucosidase Glycogen

MPS I IDUA Iduronidase Dermatin sulfate, Heparin sulfate

MPS II IDS Iduronate sulfatase Dermatin sulfate, Heparin sulfate

MPS III A SGSH N-Sulfoglucosamine sulfohydrolase Heparan sulfate

MPS III B NAGLU -N-Acetylglucosaminidase Heparan sulfate

MPS III C HGSNAT Heparan--glucosaminide N-acetyltransferase Heparan sulfate

MPS III D GNS Glucosamine (N-acetyl)-6-sulfatase Heparan sulfate

MPS IVA GALNS N-Acetylglucosamine 6 sulfatase Keratan sulfate, Chontroitin sulfate, Keratin sulfate

MPS IV B GALB1 -Galactosidase GM1-ganglioside and Keratan sulfate

MPS VI ARSB N-Acetylgalactosamine 4-Sulfatase Dermatan sulfate

MPS VII GUSB -Glucoronidase Chondroitin sulfate

Niemann-Pick A-B SMPD1 Acid sphingomyelin phosphodiesterase 1 Sphingomyelin

Niemann-Pick C NPC1-2 NPC-1 and NPC-2 Cholesterol, lipids

Infantile NCL CLN1(PPT1) Palmitoyl-protein thioesterase 1 Lipopigments (CLN1)

Late infantile NCL (CLN2) CLN2 (TTP1) Tripeptiyl-peptidase 1 Lipopigments

MLD ARSA Arylsulfatase A Sulfatides

Most of the lysosomal enzymes involved in LSDs are of donor cells to the organism in order to obtain significant glycoproteins that are synthesized in the endoplasmic reticu- therapeutic effects Fig. (1) [5]. lum and are transported via Golgi to the lysosome, although The aim of this review is to describe recent patents and a number of molecules are secreted from the cell. This pro- new strategic approaches for enzyme delivery to highlight portion of secreted enzyme is available for recapture by the the most relevant aspects, concerning next generation LSDs same cell or neighboring cells that internalize and deliver the treatment. protein into the lysosome. This characteristic of most of the LSDs has allowed the development of therapeutic strategies ENZYME REPLACEMENT THERAPY based on the exogenous administration of the enzyme (native or recombinant) to the cells. Moreover, newer approaches Enzyme replacement therapy (ERT) is based on the in- include the use of small molecules to enhance enzyme fold- travenous infusion of a recombinant wild type enzyme, ing and stabilization of the active site (pharmacological which is taken up into the cell through membrane receptors chaperon therapy) or to reduce the substrate synthesis [4]. (typically the mannose-6-phosphate receptor) and replaces Gene delivery and cell based therapy are also being evalu- the catalytic action of the mutated lysosomal enzyme [6, 7]. ERT is available since 1996 [8] and is the sole kind of treat- ated for LSDs therapy in a considerable number of clinical ment approved for LSD patients, with the only exception of trials. These two approaches, aim to provide enough func- a drug based on substrate reduction therapy (SRT) [9]. Effi- tional enzymes at surrounding tissues, through infection of a cacy and safety of ERT have been confirmed by extensive few cells with viral vectors or by transfering a small amount Lysosomal Storage Diseases Treatment Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 11

Lysosome B Stem cell

Golgi

D Differentiated cell

Substrate Synthetase Inhibitor Viral Vector C Active enzyme GthGrowth ftfactor Mutated enzyme

E Chaperon

Fig. (1). Schematic view of the possible approaches for LSDs treatment LSDs treatment can be approached through 5 different strategies: Gene therapy (A); Cell based therapy (B); ERT (C); SRT (D) and PC use (E). In A, transgenic viral vectors encoding a functional enzyme are injected to the patients to infect target cells. Once in the nucleus, viral DNA is transcribed and the functional enzyme is expressed in the endoplasmic reticulum (ER) and eventually traffics to the lysosome. In B, HSC expressing functional enzyme are differentiated and injected to the patient. The enzyme secreted by stem cells, together with modulat- ing factors, is taken up by patient’s cells through MP6R. In C, recombinant enzyme is directly infused in patient’s bloodstream or CSF and taken up by cells trough the MP6R, here it traffics to the lysosome and hydrolyzes accumulated substrates. In D, an inhibitor of the enzyme that synthesizes the substrate accumulated in patient cells is administrated to patient and carried over by the cell, where it is able to reduce the synthesis of the storage material. In E, a PC binds the active site of a miss-folded enzyme at the ER and facilitates its transport to the lysosome, where the chaperone is released due to low pH. Once in the lysosome, the mutated enzyme can partially perform its catalytic function. clinical trials, nevertheless almost two decades of its use Glu), also called glucocerebrosidase, which removes the highlighted important limitations for these drugs to actually linked glucose. All the three ERT compounds are recombi- achieve remission of the pathology. Up to date ERT is avail- nant forms of -Glu. A deficiency of this enzyme leads to able worldwide for 6 LSDs, including Gaucher disease (GD), accumulation of glycosphingolipids in macrophages of liver, Fabry disease (FD), Pompe disease (PD) and Mucopolysac- bone marrow and spleen. Therefore, patients with GD ex- charidosis (MPS) types I, II, and VI (Table 2). hibit hematological manifestations such as anemia thrombo- cytopenia, hepatosplenomegaly, skeletal deformation and Imiglucerase (Cerazyme, Genzyme) was the first recom- also neurological impairment [13]. While over 200 mutations binant enzyme for LSD therapy to be introduced in the mar- were identified for GD, 80% of the patients carries either the ket for Gaucher disease (GD) treatment [10] and was pro- N370S mutation or the L444P mutation, in at least one of the duced in Chinese Hamster Ovary cell line (CHO). This product was followed by Velaglucerase alfa (Vpriv, Shire alleles. The N370S is usually associated with Type I GD which is related to a milder phenotype, due to prevalent ac- Inc.), produced in a human cell line using gene activation cumulation of substrate in liver and spleen, while the L444P technology [11] and TaligluceraseAlfa, produced in a plant mutation causes Type III GD, a more severe condition char- cell line (Elelyso, Pfizer) [12]. In GD, the most prevalent acterized for central nervous system (CNS) involvement [14, LSD, glucosylceramide is metabolized by -glucosidase (- 15]. Treatment with ERT, is generally associated with 12 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 Ortolano et al.

Table 2. Available ERT for LSDs.

DISEASE KIND OF THERAPY ACTIVE COMPOUND PATENT

Gaucher

ERT Imiglucerase US5549892-1996

ERT Velaglucerase alfa US20110027254

ERT TaligluceraseAlfa US2008038232

SRT Miglustat US5525616

Fabry

ERT Agalsidase alfa WO19989811206

ERT Agalsidase beta US5356804-1994

Mygalstat US20020035072 PC (Clinical trial phase III not approved) US20040242539

Pompe

ERT Glucosidase alfa WO0205841

MPS I

ERT –L-Iduronidase US6426208

MPS II

ERT Idursulfase US5932211

MPS VII

ERT Galsulfase WO2004043373

Infantile NCL (CLN2)

ERT in CSF Rh Tripeptidyl peptidase 1 WO2007084737 (BMN190) significant improvement in organ volume, anemia, thrombocy- century afterwards [19]. Dysfunction of -Gal A, encoded by topenia and plasma levels of biomarkers. Long-term analysis GLA gene, causes globotrialosylceramide (Gb3) accumula- of the safety of imiglucerase therapy demonstrates a low rate tion in the lysosomes of different cells, especially dermal of adverse events and sero-conversion. The majority of fre- fibroblasts, cardiomyocytes vascular endothelial cells and quently reported adverse events related to imiglucerase were perineural cells Fig. (2). Due to its multisystemic involve- infusion-associated reactions which did not require discon- ment FD, may present a wide variety of clinical manifesta- tinuation of treatment [16]. The success of ERT in GD is tions, although common symptoms are acroparesthesia, an- strictly related to the fact that macrophages are the main target giokeratomas, neuropathic pain, cardiomyopathy, renal fail- for the enzyme and they can be efficiently accessed through ure and stroke. FD is an X- linked disease historically intravenous injection. However, the active compounds used thought to affect only men. Nowadays, it is understood that are not able to cross the blood brain barrier (BBB) and there- females are also affected because of the random inactivation fore reach the CNS, so that their efficacy in GD type III is of one of the X chromosomes so that it is possible that they limited. develop the disease with severe manifestations [20]. Trials performed with either agalsidase alfa or beta showed similar The first ERT treatment for Fabry Disease (FD) agalsi- rates of safety and efficacy for both medicaments and life- dase beta (Fabrazyme, Genzyme) [17] was approved ap- threatening side effects were absent. ERT treatment signifi- proximately 10 years ago and quickly followed by the devel- cantly reduced Gb3 concentration in heart and kidney and opment of agalsidase alfa (Replagal, Shire Inc.) [18], which also improved pain-related quality of life in patients up to six is available in Europe. Both active compound are recombinant forms of -galactosidase A (-Gal A), the enzyme months of treatment [21]. which is compromised in FD, and differs only for their gly- ERT treatment for PD is performed with glucosidase alfa cosilation pattern, since agalsidase beta is produced in CHO (Myozime, Genzyme) [22]. PD is determined by a deficit of cells, while agalsidase alfa is produced in a human cell line acid -Glucosidase (-Glu), an hydrolase responsible for the (clone RAG 001). FD was originally described in 1890, al- cleavage of the -1,4- and -1,6-glycosidic bonds of glyco- though the genetic cause of the disorder was detected over a gen, whose accumulation causes cellular dysfunction in mul- Lysosomal Storage Diseases Treatment Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 13

Fig. (2). Immunofluorescence of Gb3 in skin biopsy of an FD patient. Gb3 deposit is highly marked in fibroblast and the fluorescence pattern is compatible with intralysosomal localization tiple tissues, especially affecting skeletal and cardiac mus- disease, whose patients usually die within 2-3 years, courses cles. The infantile onset of PD is characterized by general- with osteo-articular involvement, skeletal deformations, he- ized muscle weakness, hypotonia, hepatomegaly and hyper- pato-splenomegaly, spinal cord compression due to malfor- trophic cardiomyopathy that lead to death, typically during mation of the occipito-cervical vertebra, respiratory- the first year of life due to cardio-respiratory failure. Late obstructive-pathology and mental delay [32]. ERT with onset forms of PD feature a progressive muscle weakness idursulfatase ameliorate some clinical parameters and gen- without significant cardiomyopathy [23]. eral life quality, however the drug is more efficient if treat- ERT has been shown to reduce cardiac pathology and ment starts before the manifestation of neurologic symptoms, delay the need for ventilator support in the infantile form of against which the drug does not have significant effects, the disease; however variable effects of the drug were re- since it can not cross the BBB. MPSVI is due to a deficit of corded for adult patients presenting mainly skeletal muscle N-acetylgalactosamine-4-sulfatase, also called arylsulfatase involvement [24]. The explanation for this variability is un- B, which is responsible for the catabolism of dermatan sul- clear and contributing factors may include the age of onset of fate. Neurologic impairment is absent in MPSVI patients, the treatment, the extent of pre-existing damage, immune due to lack of heparan sulfate accumulation and therefore responses, rapid preclearance of the recombinant enzyme these patients usually survive until adulthood [33]. Galsulfase and poor accessibility to motor neurons [25, 26]. (Naglazyme, Genzyme) [34] is well tolerated and determines a significant reduction in urinary excretion of dermatan sulfate ERT has been also approved for three types of muco- and a significant improvement in walking test [35]. polysaccharidosis, LSDs caused by the alteration of enzymes which determine the accumulation of glycosaminoglycans During the last decade new expression systems have been (GAGs). MPS I is related to a defect in -L-iduronidase, developed in order to increase production yield and reduce causing accumulation of GAGs such as heparan sulfate and costs of ERT [36]. dermatan sulfate. The clinical spectra of MPS I is very Calhoun et al. [37] developed a system based on Bacu- broad, including a very severe form, known as Hurler dis- lovirus expression systems, which allow high yield produc- ease, characterized by pediatric onset, facial coarsening, car- tion of recombinant –Gal A in insect ovary cell line from dio-respiratory system impairment and neurologic involve- Spodoptera frugiperda (Sf9). Recombinant enzyme was cap- ment. However, MPS I may course with a milder clinical tured by Fabry patient‘s derived fibroblast allowing 59% phenotype, known as Scheie disease, generally diagnosed increment in enzyme concentration and complete activity during adulthood and manifesting signs, such as delayed restoration. Similar results were found by Berent et al. for growth, scoliosis and joint stiffness. The intermediate condi- the -Glu expressed in Bm5, Sf21 and High Five cells [38]. tion, which is typically treated with ERT, is defined as Hurler-Scheie disease [27]. Treatment of MPS I with –L- Fogher and Reggi [39] expressed human -glu, -glu, iduronidase (Aldurazyme, Genzyme) [28] has shown im- and -gal A in plant Nicotina tabacum cultures, achieving a provement in skeleton size and reduction of hepatomegaly, yield of about 1.5% of total protein content from tobacco although the drug is not able to cross the BBB [29, 30]. seeds. Similarly, Pogue et al. presented an invention in Idursulfase (Elaprase, Shire Inc.) [31] is used for the treat- which tobacco plants expressed -Glu or -Gal A obtaining ment of MPSII or Hunter syndrome, an X-linked inherited typically 435000 units of enzyme from 1kg of tobacco, disease due to the impairment of iduronate-2-sulfate. The which could be increased up to 140-160 milions units using 14 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 Ortolano et al. young actively growing transgenic Nicotina benthamiana target the recombinant enzyme in cellular types that do not [40]. Using transgenic tobacco, Radin et al. [41] described express the mannose receptor, since it is based on the insu- the production of native human -Gly and -L-iduronidase lin-like growth factor/cation-indipendent Mannose-6- with appropriate co-translational and post-translational Phosphate Receptor M6PR (IGF-II/CI_MPR). Aerts et al. modifications. [48] used a -Gal A substrate analogue like ceramidetri- hexoside-sphingosine prior to intravenous administration of A different strategy was used by Reuser et al. [42] who the recombinant enzyme to bind catalytic site and prevent the developed transgenic mice secreting -Glu in their milk to enzyme from interaction with chemical structure in the blood obtain a more human-like glycosylation pattern. The pro- stream resulting in undesired retention and reduced targeting. duced enzyme was active in Pompe patient’s derived fibro- The substrate also stabilizes the active site and prevent from blasts at levels similar to those obtained with the recombi- non physiological enzyme catalyzed reactions in the circula- nant enzymes produced in CHO and other cellular systems tion. Arranz [49] chemically fused -Glu with a cell pene- used in production of approved drugs. Experiments per- trating peptide which facilitates active transport through bio- formed in PD knock out mice showed an increment in -Glu logical membranes into the lysosome. Conjugation with activity in the liver, spleen, heart and skeletal muscle. Clini- DPV3 peptide did not affect enzyme activity and is taken up cal trials (phase I/II) showed reduction in glycogen storages into MPS VII fibroblasts, significantly improving degrada- in animals receiving biweekly injections of the enzyme [43]. tion of intracellular GAGs. In spite of the partial amelioration of patient’s quality of Nevertheless, the most important limitation for ERT re- life determined by the use of ERT, all of these highly expen- mains its poor capacity to specifically target deep layers of sive treatments, failed to meet the expectations in terms of many affected organs, including bone and brain, affected in efficacy and none of them is active against neurologic mani- the most severe phenotypes. The development of new strate- festations. A key factor to improve the effect of these thera- gies to effectively target the brain and bone is a primary is- pies is the increase of enzyme bioavailability by controlling sue to improve quality of life for patients with LSDs. Ex- immune reactions against therapeutic enzymes and other periments in tissue culture and in animal models have shown degradation processes which may occur in the bloodstream. increased efficiency of enzyme delivery for second- Indeed a negative impact of antibody formation on therapeu- generation products and novel delivery systems are now un- tic effects has been reported for GD, FD, MPS type I, and der experimentation [50]. Neurologic symptoms represent PD, for which also autophagic build up was described [36]. some of the most significant manifestation of LSDs in terms Vaccaro et al. [44] used modified protozoan organisms de- of mortality. Indeed cerebrovascular events such as stroke rived from Leishmania trypanosome strains expressing - are a major risk factor for premature death in patients with Glu, -Gal, or hexosaminidase A, B, S. Modified protozoan FD and CNS involvement is the worst consequence of MPSs cultures could be used to purify the recombinant enzyme and GD type III among other LSDs. from cell or cell supernatant, but also, and more interest- ingly, for direct administration of the protozoan cells and To improve delivery to the CNS, intrathecal routes of in situ production of the enzyme (Bactofection), thus in- administration have been explored. Salamant-Miller et al. creasing local availability of the enzyme. In spite of the [51] provided an effective method to distribute recombinant therapeutic potential of this approach, preclinical trials have enzymes to the CNS by intrathecal delivery, directly ac- to be carried out in order to check the safety of the treatment cessing the spinal canal through a lumbar injection of the and the possible virulence of the protozoan organism. medicament. The injection into the cerebrospinal fluid allows the enzyme to reach different brain areas. In particu- Recent works were also developed to improve the lar, this method is meant to be used in the treatment of amount of enzyme delivered to tissues by altering either the LSDs such as Krabb disease, Niemann-Pick Adisease and ligand or the M6PR expression on target cells, in the attempt MPS I, II, IIIA. to target organs with a key role in LSD physiopathology [45]. Recently, ERT have also been developed for the treatment of Late Infantile Neuronal Ceroid Lipofuscinosis Sly et al. [46] inactivated chemically the protein oligo- (NCL) [52, 53]. saccharides by treating the enzyme sequentially with sodium metaperiodate and sodium borohydrate. This treatment inac- NCL is the general name for a family of at least eight tivates the two traditional recognition markers that mediate genetically separate neurodegenerative disorders that result rapid clearance from the blood by means of the mannose and from excessive accumulation of lipofuscin in tissues. These mannose6-phosphate receptors. This increased the half life in are autosomal recessive disorders of the CNS with general circulation from 11 minutes to 18.5h. Efficacy of this en- fatal course which typically begin during childhood. NCLs zyme was determined in MPS VII mouse model. After 12 have been classified and named referring to the affected weeks of treatment remarkable clearance of the storage ma- genes. Up to date, linked different genes determining NCLs terials in cortical neurons and visceral organs of mice was have been identified: CLN1 (PPT1), CLN2 (or TPP1), CLN3, observed. CLN4, CLN5, CLN6, CLN7 (MFSD8), CLN8, CLN10 (CTSD) [54]. Martini et al. Concino M [47] patented polypeptide se- quences and polypeptide linkers to be potentially used in San ERT for CLN2 is based on the recombinant form of Filippo (MPSIII B) treatment. The polypeptide linker is Tripeptidyl-peptidase 1, encoded by the TTP1 gene and made up of sequential repeats of the following sequence identified as BMN190. Biomarin started a phase1/2 clinical GAPGGGGGAAAAAGGGGG. This strategy is useful to trial (NCT01907087) to treat CLN2 by direct injection of Lysosomal Storage Diseases Treatment Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 15

BMN190 in the cerebrospinal fluid using an intracranial of molecules preapproved for different medical indications device. (Table 3). All intra-cerebrospinal fluid ERT studies performed in Sandhoff disease (SD) and Tay-Sachs disease (TSD) are mice resulted in improved neurologic function and reduction LSDs involving significant neurologic manifestations and of neuropathological lesions has also been described in the therefore ERT is not an adequate therapy. PC can be used to brain of dog models (MPS I, MPS IIIA) [55]. However, sev- reach the CNS and increase lysosomal translocation of mutant eral questions remain regarding this approach, in particular -hexosaminidase (-Hex) subunits to provide an effective which route of injection (ventricular, cisternal or lumbar) therapy for both disorders [59]. -Hex is a dimeric enzyme provides the most widespread distribution of the enzyme in that is required for the removal of the terminal N- brain parenchyma; which type of enzyme delivery, bolus or acetylgalactosamine from several glycosphingolipids (GSLs), sustained, is preferable and, finally, what frequency of ad- as well as from some glycoproteins and oligosaccharides. ministration has to be applied. The intrathecal injection ap- -Hex can be formed by an and a subunit (-Hex A, het- proach is currently being tested in clinical trials for MPSs. erodimer) or by two or subunits (-Hex S and -Hex B (MPS I; NCT00638547 and NCT00852358; MPS II, homodimer, respectively). Genetic mutations in the -subunit NCT00920647 and MPS IIIA; NCT01155778) [56]. Another resulted in compromised activity of both -Hex A and -Hex approach to cross the BBB was developed by Fogh et al. B leading to the accumulation of GSLs, GAGs, acetylgalacto- [56], who produced recombinant -mannosidase fused at 5 samine and N-acetylglucosamine, which are responsible for end to the diphteria toxin translocation domain. Human fi- SD. On the other end, TSD results from mutations in the - broblasts were able to efficiently uptake the recombinant subunit, leading to -Hex A deficiency and the accumulation enzyme, although in vivo evaluation was not carried out. of ganglioside GM-2 as major storage product, even though On the other end, Stern et al. [57] produced chimeric acetylgalactosamine and N-acetylglucosamine are also accu- proteins comprising a polypeptide sequence selected from mulated [60]. leptin or granulocyte-colony-stimulating-factor (GCSf), Mahuran et al. developed a series of carbohydrates as which can cross the BBB. They generated GCSf-- possible PC agents for -Hex [61]. In particular, they identi- hexosaminidase A and leptin -hexosidase conjugates to fied the iminosugars acetamidodeoxynojirimycin, acetami- intravenously infused Tay-Sachs disease mouse models with dodideoxynojirimycin and a related acetoamido analogue of the GCSf. Mice showed partially recovered enzyme activity castanospermine as potent inhibitors of -Hex. These com- in the brain. pounds were found to increase enzymatic activity approxi- Finally, Boivin et al. [58] produced recombinant iduro- mately 2.5 fold in TSD patient-derived fibroblasts and the nate-2-sulfate as a fusion protein with Angiopep-2, specific enzymatic levels were sustained for 24-48 h after drug re- peptidic moieties (Lys-Arg-X3-X4-X5-Lys), which allow the moval [59]. recombinant enzyme to cross the BBB and increase its activ- HTS was performed to identify 24 initial hits among ity in the lysosome compared to wild type enzyme. This 50000 compounds; the most significant of them resulted to molecule may be potentially used for the treatment of MPS be pyrimethamine, an approved antimalarial agent [62]. type II. Pyrimethamine significantly increases enzymatic activity in all the -subunit mutants evaluated, however it determines PHARMACOLOGICAL CHAPERONES an insufficient increase in enzymatic activity of the -subunit mutants tested. In spite of this limitation, a clinical trial LSDs treatment can be approached through the use of Phase1/2 was initiated in patients with SD or TSD. The 8 small molecules which stabilize affected enzyme folding treated patients showed significant enzymatic activity in- (pharmacological chaperones) or reduce substrate synthesis. crease, however severe side effects, such as ataxia or blurred A pharmacological chaperone (PC) is a specific inhibitor vision, among others, appeared during the trial, causing sus- that favors the correct folding of a mutated enzyme, avoiding pension of the study [63]. its degradation in the endoplasmic reticulum and enhancing PC therapy for FD was mainly developed using mole- its activity. In LSDs treatment, the PC stabilizes the tertiary cules mimicking the structure of D-galactose. Fan et al. [64- structure of the lysosomal enzyme, allowing it to traffic 66] described the use of 1-deoxygalactonojirimycin (DGJ), through the secretory pathway and reach the lysosome. Here as PC acting on -Gal A. Exposure to DGJ (0.2-20 μM) of a catalytically competent enzyme is released upon acid hy- primary lymphoblast cultures from FD patients, caused a drolysis of the PC and maintained in the active conformation concentration dependent increase of -Gal A activity which for an extended period of time [4]. was maintained for 5 days. The chaperoning action on the Since the majority of PCs identified binds to the catalytic mutated enzyme depends on the interaction of the NH group site of the enzyme, substrate turnover is favored by high sub- of the iminosugar with the carboxylate of the aspartic acid strate concentration and by binders that present shorter half- 170 in the active site of the enzyme, as shown by crystallog- life than the mutant enzyme as well as reduced binding affin- raphy [67]. ity at lysosomal pH. In vivo studies with DGJ on FD transgenic mouse models PCs for LSDs treatment have been designed and synthe- showed significant increase of -Gal A activity in heart, kid- sized based on the structure of the natural substrate or alter- ney, spleen and liver of the treated animals, as well as de- natively identified through high throughput screening (HTS) creased levels of Gb3 in the affected tissues [68]. DJG is

16 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 Ortolano et al.

Table 3. PCs to be used in LSD Treatment.

DISEASE COMPOUND PATENT

Gaucher OH

HO OH

WO2004037373 OH N

8 OH

HO OH

WO2013075227 OH US20130184352 N

8 OH

HO OH

US20030119874 OH N

8 OH

HO OH US5844102

N H

N N WO2012078855

NH O

O O

O S

N O

N WO2009049422

N S

Lysosomal Storage Diseases Treatment Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 17

Table (1) contd….

DISEASE COMPOUND PATENT

H N OH Tay Sachs WO2004103368 Shandon O OH X N H OH

HO OH WO2006125141 Pompe WO2007150064 OH N H

OH OH

HO OH HO OH US20020035072 Fabry US20040242539 OH OH N N H H

O R3 N R2 Newmann-Pick WO2005051331 O P O (CH2)m

Rn HO

O OH

Krabbe WO2009061906 N

HO OH

OH N Gangliosidosis WO2008034575

H N O

O currently the active component of mygalstat hydrochloride, a Sawkar [71, 72] and co-workers found that N-(n- new potential drug that is currently being evaluated for nonyl)deoxynojirimycin can increase -Glu activity in cell treatment of FD in phase 3 clinical trials [69]. based assays acting as a PC in GD therapy. This molecule allows a 1.65 fold increase of enzyme activity in homozy- Patent by Elfrida and Do [70] describes a method to predict the response to a PC-based treatment of FD. In particu- gous cells carrying the N370S mutation, persisting during 6 days. Nevertheless, the same compound did not affect -Glu lar, the invention provides an in vitro assay for determining activity in cells derived from subject carrying the homozy- -Gal A responsiveness to a PC such as 1-deoxygalactono- gous mutation L444P, associated with GD’s most severe jirimycin in a cell line expressing a mutant form -Gal A, the phenotype. Following N-(n-nonyl)deoxynojirimycin many same method is potentially applicable as diagnosis method other iminosugars analogues were developed and tested on for FD patients. 18 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 Ortolano et al. patient’s derived cells: among them 1-adamantylcarboxamide pathologies. Suetz et al. [87, 88] synthesized a series of was able to increase enzyme activity in N370S and G202R DGJ-analogs, which were tested in Gangliosidosis patient mutants, but not in L444P. derived fibroblasts, however these compound were little ef- N,N-Disubstituted carboxamide chaperones were pat- fective against the most severe forms of the disease [89]. ented by Mahuran et al. and resulted in 1.6 fold increased A possible PC therapy for Krabbe disease was developed activity for -glucosidase in N370S affected patient’s de- by Lee et al. [90]. Krabbe disease or globoid cell leukodys- rived fibroblast [73-75]. trophy is caused by low galactocerebrosidase activity result- Yu et al. [76, 77] designed -1-C-alkyl iminosugars to ing in the accumulation of the highly toxic GSL Galacto- act as PCs in GD and found about 2 fold increased -Glu sylsphingosine. The disease is characterized by severe de- activity, following treatment of N370S mutant cells with one myelization with apoptosis of oligodendrocytes, since Galac- of the synthesized analogues. The same compound also de- tosylsphingosine is a major component of white matter, termined a modest increase (1.3 fold) in enzyme activity in which is also metabolized by galactocerebrosidase. The con- cell derived from patients carrying the in L444P mutation. sequence of this is a profound neurodegeneration and premature death. Following HTS, Loboline was identified as a Fan et al. [78, 79] identified isofagamin as a promising weak inhibitor of galactocerebrosidase in D528N mutant cell lead structure for the treatment of GD and were able to show lines, showing a 50% increase in enzyme activity. However, increased -Glu activity in cells derived from N370S and this compound presented significant BBB penetration and L444P affected patients. The tested azasugar causes a con- can be considered as an optimal leading molecule for chap- formational change in the enzyme revealing a hydrophobic eroning therapy [91]. groove postulated to interact with the ceramide moiety of the natural substrate [80]. The obtained results were confirmed Okumiya et al. [92-94] tested 1-deoxynojirimycin and following treatment of mutant L444P mice and put the basis several N-alkylated analogues in cultured fibroblast cell lines for Phase 1 and 2 clinical trials, which unfortunately were from PD patients. These iminosugars are 10x to 250x more afterwards discontinued, failing to show a clinically meaning selective for -Glu compared to -Glu form. In details, 1- full end point in most of the involved patients. deoxynojirimycin showed increased activity in 2 out of 5 tested cell lines. Cell lines derived from patients carrying It is worth noting that the biological activity of the de- many different mutations (ej.L552P, S529V, A445P, R432C) scribed compounds in GD treatment is due not only to the were tested and give satisfactory results in chaperoning ac- carbohydrate mimetic portion of the molecule interacting tivity for -Glu [95-97]. Preliminary studied of 1- with the active site, but also to the contribution of the long deoxynojirimycin administration to PD mouse model alkyl chains mimicking the ceramide portion of the substrate showed daily important reduction of substrate in heart, dia- and helping to cross cell membranes. phragm, gastrocnemius, soleus and brain [98]. Compound Maegawa et al. [81] performed a HTS of FDA approved AT2220 is being evaluated in a phase 2 clinical trials and at compounds to find good PC candidates for GD therapy. lower doses to avoid toxicity. Among the analyzed molecules, ambroxole was able to sta- Schuchman and Desnick [99] patented a molecular chap- bilize N370S mutated -Glu. Activity was increased in pa- eron of potential use in Niemann-Pick disease type A and B tient’s derived cells homozygous for the N370S mutation. In to improve activity of sphingomyelinase. The lysosomal an extensive HST study designed with the goal to find mole- storage disorders Niemann-Pick disease, SMPD1-associated cule increasing traslocation to the lysosome, Marugan et al. (Type A and B) are characterized by deficiency in acid [82] found quinazoline compounds able to stabilize -Glu. sphingomyelinase, caused by a mutation in the SMPD1 gene. At the same time they identified pyrazolopyrimidine as a Mutations to this gene are more commonly found in those of class of non-inhibitory compounds which can stabilize the Ashkenazi Jewish descent (1:80-1:100) or of North African mutated enzyme and improve its trafficking. These mole- descent. Niemann-Pick Type C (NPC) is also a lysosomal cules are of particular interest for their ability to cross the storage disorder, but instead is caused by mutations in either BBB [83, 84]. NPC1 or NPC2 gene. These PCs are based on the structure of sphingomielin, ceramide or phosphonucleotide analogues Finally, an inhibition-based primary screen identified [100]. 5[(4-methyl-phenyl)thio]quinazoline-2,4-diamine [85] which demonstrated a 2.5 fold increased -glu activity when used at SUBSTRATE REDUCTION THERAPY 12.5μM on cells derived from patient carrying the N370S Substrate reduction therapy (SRT) consists in the use of mutation [86]. This compound is also active to stabilize - an inhibitor for an enzyme which is involved in the synthesis galactosidase (-Gal) and can be potentially used also in PC of the substrate for the affected enzyme. At present the only therapy of Gangliosidosis and Morquio B disease (MPS IV SRT approved for GD is based on N-butyldeoxynojirimycin B), which are caused by a deficit of this enzyme. Compro- (Miglustat) administration Fig. (3) [101]. This active princi- mised -Gal activity in gangliosoidosis leads to lysosomal ple inhibits glucosylceramide synthase avoiding glycosphin- accumulation of glycolipids associated to severe neurode- golipids accumulation. Miglustat may only be used in the generation. On the other end, Morquio B disease mostly af- treatment of GD type 1 patients for whom enzyme replace- fects skeletal system and has little if any neurological in- ment therapy is unsuitable and it has been approved in the volvement. The different phenotypes in the two diseases European Union for the treatment of progressive neurologi- seems to be mutation related. Iminosuguras derivates of ga- cal manifestations in adult or pediatric patients with Nie- lactose were also considered for the PC treatment of these mann-Pick type C disease (NPC) [102]. Lysosomal Storage Diseases Treatment Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 19

OH also in clinical trials, such as the one for metachromatic leu- HO kodystrophy (MLD) (NCT00633139). OH MLD is autosomal recessive LSD caused by mutations in the ARSA gene and leading to the deficiency of the arylsulfa- OH tase A, which causes widespread demyelinization and neu- N rodegeneration. In 2010 clinical trials have been started test- ing lentiviral vectors to transfer ARSA gene in bone marrow derived hematopoietic stem cell. Biffi et al. found that the treatment halted disease manifestations in presymptomatic children with late-infantile MLD, who were followed during N-butyldeoxynojirimycin (Miglustat) 24 months [105, 106]. US5525616 (1996) Mucopolysaccharidosis VII (MPS VII, Sly disease) is the Fig. (3). Compound used for SRT of GD. Structure of N- inherited metabolic disorder with the highest number of trials butyldeoxynojirimycin (Miglustat), the active compound of an approved therapy for GD based on SRT. performed, exploring different alternatives to reach the CNS [107]. Davidson et al. [108] expressed the -glucoronidase STR is also used for the treatment of infantile neph- gene to intravenously infuse a feline immunodeficiency virus ropathic cystinosis, a rare genetic disease due to dysfunction (FIV)-derived retrovirus, which was capable to treat and of cystinosine transporter expressed in lysosomal membrane. prevent visual and neurologic alterations of the disease. Cysteamine is used in these patients to convert accumulated AAV-based constructs were also designed for the treat- cystine into cysteine and cysteine-cysteamine mixed disul- ment of MPSVII. AAV vectors are gene transfer tools that fide, both of which can pass through the lysosomal mem- largely contributed to the advances of biologic therapies in brane of patients with cystinosis [103]. recent years, owing to their low immunogenicity, no patho- The same active compound has also been used to treat genicity, and stability [109]. NCL related to PPT1 which is involved in lysosomal degra- Patent by Podsakoff [110] describes AAV vectors ex- dation of S-fatty acylated proteins [104]. pressing -glucuronidase for potential therapy of MPS VII. GENE THERAPY Intravenously injected animals presented long term expression of the gene and significant reduction of GAGs storage An array of different viral vectors and viral serotypes, in tissues. Moreover intrathecal injection of the same vector including lentivirus, adenovirus, and adeno-associated virus into the cerebrospinal fluid, to both neonatal and adult (AAV) has been tested in the last decades to achieve gene animals, results in therapeutic levels of -glucuronidase into therapy of LSDs and in particular to achieve remission of the brain and the complete elimination of GAGs storage in neurological manifestations (Table 4). the tissue. Promising preclinical tests were also undertaken These innovative methods for delivering recombinant in dog models of MPSI and MPS III B to access efficiency enzymes into the CNS increased in interest, following the of AAV-based gene therapies in short to medium terms successful results obtained not only in animal models, but [111-114].

Table 4. Gene and Cell Therapy Based Clinical Trials for LSDs.

Disease Vector Delivery Method Patent Clinical Trial

MLD Lentivirus Intravenus infusion WO2010125471 Phase I/II IT0019 NCT00633139

MPSVII (FIV)-Derived retrovirus Intravenus infusion WO2000073482 Phase I US07-58 (Sly disease)

MPSI Retrovirus Intraperitoneal US20040023218 FR0005

POMPE AAV1 Intramuscular US20080069803 Phase I/II NCT00976352

Late Infantile NCL AAV2 /AAVrh10 Intracraneal WO2008154198 Phase I (CLN2) NCT00151216 NCT01161576

Infantile and Late infantile NCL Neural stem cell transplantation Neurosurgical transplantation US20026497872 NCT00337636 (CLN1 and CLN2)

Niemann-Pick HSCT Bone marrow transplant US2007009500 NCT00668564

20 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 Ortolano et al.

Moreover, Podsakoff et al. registered patents [115,116] Sena-Estevez et al. [122] produced constructs that can for AAV1-mediated gene transfer into muscle tissue of GAA efficiently reach the brain of patients and are potentially use- gene, encoding acid -glucosidase, to treat PD. PD is a good ful for gene therapy of TSD, SD and GM1-gangliosidosis. candidate disease to test the intramuscular injection route of The two constructs are based on AAVrh8 and respectively administration, since the affected isoform of the enzyme is express the -subunit and -subunit of -N-acetylhexosa- mainly expressed in the muscle. Muscle delivery can be con- minidase. The active compounds are administred in didder- sidered for treatment of metabolic diseases only when low ent brain areas (i.e., thalamus, striatum, deep cerebellar nu- levels of expression are required and when there is no need clei, ventral tegmental area, and lateral ventricles) through an for secretion of the transgene. Efficiency of these constructs injection in the CSF. is being tested in an ongoing clinical trial (NCT00976352) For FD Yew et al. [123] developed recombinant viral and through a series of intra-diaphragmatic injections. Initial non viral vectors comprising a gene encoding the human safety and ventilatory outcomes of these trials were pub- lysosomal enzyme -Gal A capable of transducing the target lished this year [117]. cells and producing a sustained expression of the gene. Intraparenchymal injection was also attempted to reach Similar approaches, based on AAV vectors, were pro- global delivery of viral vectors in the brain in LSDs severely posed by Desmaris et al. for Hurler disease and MLD [124], affecting CNS such as NCL [118-120] and Niemann-Pick A as well as by Mahuran et al. for the treatment of GD [125]. disease [121]. AAV serotype 2 vector expressing the human CLN2 CELL BASED THERAPY cDNA (AAV2 CU h-CLN2) was administered to 12 loca- tions in the CNS of 10 children with late infantile NCL. Cell-based therapy has also been explored to deliver However, the treatment resulted in death in one of the pa- genes of interest in the brain and potentially treat LSD patients due to serious adverse effects, the etiology of which tients. The introduction of gene-modified or un-modified could not be determined and was not apparently related to cells into LSD patient may be undertaken in two ways: direct the vector AAV2 CU hCLN2. Assessment of the neurologic CNS implantation or engraftment outside the brain, most rating scale, which was the primary outcome variable, dem- commonly involving hematopoietic stem cell transplantation onstrated a significantly reduced rate of decline compared (HSCT). with control subjects. In a second study the vector was in- Direct engraftment of human CNS stem cells into the CNS jected in rats and monkeys to establish a safe dose to be used has been used in a completed clinical trial in infantile and late in clinical trials on human patients [54]. infantile neuronal ceroid lipofuscinosis (NCT00337636). Tri- Dodge and Cheng also [120] provided methods to deliver als demonstrated the feasibility of this approach and the ab- a gene to the CNS and affected visceral organs by intraven- sence of transplantation-related serious adverse events, tricular administration of a recombinant neurotrophic AAV which supports further exploration of human CNS stem cells based viral vector, expressing other defective enzyme related transplantation as a potential treatment for select subtypes of to LSDs. These constructs encode genes linked to alterations NCL. [126, 127]. It was also reported that immunosupres- in the CNS, such as Niemann-Pick A or B, Hurler disease, sion by mycophenolate, may be useful in slowing down the Hunter disease, Sly disease and GD. As an example, N- progress of CLN3 related NCL [128]. acetyl glucosamine-1-phosphotransferase is targeted to the Shihabuddin et al. [129] disclosed methods for treating central nervous system in subjects having Mucopolysaccha- lysosomal storage diseases that affect the CNS. The method ridoses (MPS) by the administration of recombinant AAV4 involves administering non-immortalized neural stem cells encoding this transgene. secreting lysosomal hydrolases into the brain. In particular, Patent by Passini [121] describes a suitable method for this invention claims a possible treatment for Niemann-Pick therapeutic approach of LSDs affecting the CNS, such as A disease. Niemann-Pick Type A and Type B. The method consists in Greater research focus has been spent examining the en- contacting an axonal ending of a neuron with a high titer of graftment outside the brain [130]. This therapeutic approach AAV carrying a therapeutic transgene (e.g. acid sphingo- consists in HSCT of autologous cells that have been geneti- myelinase), so that the vector is axonally transported in a cally modified to express the missing protein. Three clinical retrograde fashion and the product can be expressed distally trials are currently underway to acces the efficacy of modi- to the administration site. The invention is based in part on fied hematopoietic stem cells in MPSII, MPSVII and MLD the discovery and demonstration that the injection of acid [131]. Related to this, a recent report indicates an excellent sphingomyelinase into the diseased brain of an animal model survival rate, resolution of hepatosplenomegaly and stabili- resulted in protein expression within multiple distal sites zation of cardiovascular problems in children with MPSII consistent with the topographical organization of the projec- treated with bone marrow transplantation; however neuro- tion neurons that innervate the injection site. In particular, psychological outcomes were variables and did not show a AAV-9 has shown high transduction efficiency when deliv- convincing evidence of neurological benefit [132]. ered intracranially as a result of axonal transport. Additional Autologous HSCT has also been successfully carried out studies will be required to identify the disease specific mo- in patients with Niemann Pick C type 2, at early age (16 lecular signature of the brain endothelium in humans, which months). Follow up of patients during 5 years highlighted the may differ from those of experimental animals. Lysosomal Storage Diseases Treatment Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 21 absence of respiratory compromising and gross motor devel- On the other hand, gene therapy vectors seem to be a opmental progresses [133]. good option for complete remission of the pathology, but they presents enormous difficulties to establish safe admini- Ramaswamy [134] developed a method for transgene expression in cells by homologous recombination. This in- stration regime and it is difficult to foresee long term unde- vention involves the use of dominant-negative and loss of sired effects. Moreover some of these treatments are often associated to invasive route of administration, which does function mutations in components of the intracellular Golgi not meet patient compliance. to lysosome sorting pathway as a mean to enhance the secretion of one or more lysosomal proenzyme in somatic cells At the same time, all the proposed strategic therapies are providing a treatment for LSDs, particularly in neuronal probably really effective only if associated with early onset cells. In particular, homologous recombination may be used of the treatment and presymptomatic diagnosis, as all the to engineer therapeutically useful cells, such as glial progeni- described therapeutic tools will have little efficacy to revert tor cells, mesenchymal stem cells and astrocyte precursor preexisting tissue damage. cells, to enhance secretion of one or more lysosomal en- A feasible and possibly effective strategy to improve zymes. Gain-of-function or loss-of-function mutations in LSDs therapies can be the combination between ERT and components of the intracellular Golgi to lysosome sorting PC. Single PC administration previous to ERT can stabilize pathway are used to enhance secretion of one or more the recombinant enzyme while in the neutral pH environ- lysosomal enzymes. ment of the blood and therefore ameliorate immune response Garbuzova-Davis et al. [135] patented a method for treat- and ERT half-life facilitating cellular uptake. Successful ing a fetus or embryo suspected of having congenital condi- combination therapies have already been described for GD, tions including San Filippo's syndrome, Hunter's syndrome, PD and FD in preclinical studies [138-140] and are under Hurler's syndrome, TSD, GD, von Gierke's disease, PD, study in clinical trials for FD and PD (NCT01196871, among others. The method involves intravenous injection of NCT01380743). human umbilical cord blood cells expressing lysosomal en- Another promising approach for a new treatment could zyme to a mother carrying a fetus of embryo affected by a be the correction of the genetic defects through the implant specific inborn metabolic error. of modified HSC, which may help to overcome toxicity Finally, Van der Loo et al. [136] patented optimization problems related to viral vector based gene therapy. How- methods for a successful genetic correction of diseases, me- ever, cell based therapy has to be still considerably imple- diated by hematopoietic stem cells. This invention accesses mented and also issues related to ethical debate on the source minimum HCS chimerism and gene dosage for correction of of donor cells have to be taken into account. a hematopoietic disease, such as sickle cell anemia. The in- Even though LSDs treatment is still an unmet clinical vention further relates to a method of improving and/or cor- need, the great efforts of the scientific community testified recting one or more CNS abnormalities caused by one or by the wide number of clinical studies and patents registered more lysosomal storage disease. The invention also relates to in the last decade allow being optimist on the evolution of methods of improving titer in transfection-based bioreactor therapeutic strategies for the near future. culture production or transfection-based production systems using eukaryotic cells. CONFLICT OF INTEREST CURRENT & FUTURE DEVELOPMENTS Shire Pharmaceuticals Iberica granted a research project to CN, SO and IV collaborated in the development of this In spite of the great efforts and the achieved advances in project and CS declare no conflict of interest. the field of LSDs treatment, these diseases are still life- threatening and limiting, with respect to quality of life, for ACKNOWLEDGEMENTS many patients. ERT represented an enormous improvement for some LSDs affected individuals, but also revealed to The authors wish to thank Tania Vazquez-Santos for have numerous limitations to reach the target of pathology proof revision. remission. As an example, some studies reported that ERT Research activity of the authors is supported by grants provides only minimal improvement on cardiovascular pa- from the “Instituto de Salud Carlos III” (PI11/00842 to S.O., rameters in FD patients [137]. The main limitation associ- PI10/02628 to CN) and European Union funded projects: ated to ERT such as high cost, immunologic response and Spain-RDR (IRDiRC), BIOCAPS (FP7/REGPOT-2012- lack of efficacy to CNS represents significant challenges for 2013.1, agreement no. 316265). future development of new drugs. PC are low-cost promising tools to cross the BBB and ABBREVIATIONS improve the drug action on the CNS, however they also present challenges to face for their improvement, especially AAV = Adeno Associated Virus regarding the fact that in the majority of the cases they are BBB = Blood Brain Barrier active only for specific mutations. In addition, the effects of CHO = Chinese Hamster Ovary PC on the glycosylation pattern of the protein, which influ- ence translocation process to Golgi, endosome and CNS = Central Nervous System lysosome, have not been clarified. 22 Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 2014, Vol. 8, No. 1 Ortolano et al.

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