Encapsulated engineered cells for treating a disease or condition affecting the nervous system

Abstract

The present invention relates to genetically engineered cells secreting a protein of interest for use in treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cells are encapsulated in implantable devices. The present invention also relates to a method for treating a disease or condition affecting the nervous system in a subject in need thereof, the method comprising the step of implanting a device comprising encapsulated genetically engineered cells in the subject.

Description

ENCAPSULATED ENGINEERED CELLS FOR TREATING A DISEASE OR CONDITION AFFECTING THE NERVOUS SYSTEMFIELD OF INVENTION

[0001] The present invention relates to genetically engineered cells secreting a protein of interest for use in treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cells are encapsulated in implantable devices. The present invention also relates to a method for treating a disease or condition affecting the nervous system in a subject in need thereof, the method comprising the step of implanting a device comprising encapsulated genetically engineered cells in the subject.BACKGROUND OF INVENTION

[0002] Lysosomal storage diseases (LSDs) are a group of disorders characterized by the accumulation of substrates in excess in various organs, such as, for example, the nervous system, due to the defective function of lysosomes.

[0003] Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an inherited deficiency of arylsulfatase A (ARSA) and characterized by accumulation of sulfatides in both glial cells and neurons resulting from myelin degeneration in the central and peripheral nervous systems. MLD is classified in three forms based on the age of symptom onset. Late infantile MLD is the most frequent and severe form and death usually occurs about 2 to 6 years after diagnosis. Currently, there are no available therapies to stop or delay disease progression once patients are symptomatic.

[0004] Libmeldy© medicine, an ex vivo gene therapy consisting of autologous hematopoietic stem cell transplantation of CD34 cells corrected with a lentiviral vector overexpressing human ARSA (HSCT-GT; hARSA), is the gold standard for pre-symptomatic MLD patients. However, this treatment is not efficient in symptomatic patients.

[0005] Intrathecal enzyme replacement therapy (IT-ERT) enables to deliver ARSA in the CNS. Results of a phase I-II trial, performed in early symptomatic late infantile MLD patients, confirmed a good safety profile and, for patients receiving the highest dose (100 mg every 2 weeks), a normalization of the sulfatide content in the cerebrospinal fluid (CSF) has been showed as well as a trend to a lesser decline in motor functions over time despite degradation. However, no data are available regarding the efficacy of such treatment for more severe patients and most importantly, this approach requires weekly chronic injection in hospital.

[0006] Thus, there is a need to provide treatments for MLD patients, and especially, symptomatic MLD patients, which are efficient and can improve the quality of life of patients.

[0007] The Inventors herein provide data showing that a cell-based implantable device delivering ARSA is able to correct sulfatide storage and to significantly improve neuroinflammation in a mouse model of MLD, and that a cell-based implantable device can deliver ARSA in non-human primates.

[0008] Altogether, these data show that a cell-based implantable device is able to robustly deliver a protein of interest in the brain, which supports the use of cell-based therapy with implantable devices for treating diseases or conditions affecting the nervous system described herein.SUMMARY

[0009] The present invention relates to a genetically engineered cell secreting a protein of interest for use in treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cell is encapsulated in an implantable device and wherein said disease or condition affecting the nervous system is either:i) a storage disease, such as a lysosomal storage disease, and the protein of interest is the wild-type version of the mutated protein causing the storage disease, or ii) the disease or condition is Friedreich ataxia, and the protein of interest is frataxin.

[0010] In one embodiment, said disease or condition and said protein of interest are one of the followings combinations: i) the disease or condition is metachromatic leukodystrophy (MLD), and the protein of interest is arylsulfatase A (ARSA); ii) the disease or condition is a mucopolysaccharidosis and the protein of interest is an enzyme involved in the degradation of glycosaminoglycans, preferably the disease or condition is a mucopolysaccharidosis (MPS) type III, in particular either : a. MPS type III B, and the protein of interest is N-acetyl-alpha- glucosaminidase (NAGLU); or b. MPS type III A, and the protein of interest is N-sulfoglucosamine sulfohydrolase (SGSH) iii) the disease or condition is Friedreich ataxia, and the protein of interest is frataxin.

[0011] In one embodiment, the disease or condition is MPS type III B, and the protein of interest is N-acetyl-alpha-glucosaminidase.

[0012] In one embodiment, the disease or condition is MPS type III A, and the protein of interest is N-sulfoglucosamine sulfohydrolase (SGSH).

[0013] In one embodiment, the disease or condition is Friedreich ataxia, and the protein of interest is frataxin.

[0014] In one embodiment, the protein of interest is fused with one or several peptide(s) promoting protein secretion, protein recapture and / or protein addressing to cellular compartments.

[0015] In one embodiment, the disease or condition is metachromatic leukodystrophy (MLD) and the protein of interest is ARSA, preferably human ARSA.

[0016] In one embodiment, ARSA is fused to one or several peptide(s) promoting protein secretion, protein recapture and / or protein addressing selected from the group comprising or consisting of a protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep-2 peptide (Ang-2), receptorbinding domains of human apolipoprotein B (ApoB) and ApoE, including ApoE-I and ApoE-II, a native secreted alkaline phosphatase (SEAP) secretion peptide, signal peptides derived from interleukins, and variants thereof, preferably ARSA is fused to a receptorbinding domain of ApoE II.

[0017] In one embodiment, the subject is symptomatic. In one embodiment, the symptomatic subject presents with sulfatide accumulation and neuroinflammation.

[0018] In one embodiment, the subj ect has received or is to be received a transplantation of lentiviral corrected hematopoietic stem cells secreting the protein of interest in the brain.

[0019] In one embodiment, the genetically engineered cell is an immortalized human myoblast cell or a progeny thereof derived from primary human myoblast cells.

[0020] In one embodiment, the genetically engineered cell is a cell from the immortalized human myoblast cell line deposited with CCOS under accession number 1902.

[0021] In one embodiment, the implantable device is an implantable capsule.

[0022] In one embodiment, the implantable capsule comprises a cell receiving portion comprising a porous membrane surrounding a cell receiving chamber for receiving immortalized cells in a liquid media, and a cell support matrix comprising at least one yam inserted within the cell receiving chamber configured for the arrangement of the immortalized cells within the cell receiving chamber.

[0023] In one embodiment, the implantable device comprises from about 100 000 to about 2 million of cells, preferably from about 150 000 to about 1.5 million of cells, more preferably from about 250 000 to about 1 million of cells.

[0024] In one embodiment, the implantable device is adapted to be implanted subcutaneously, intramuscularly, intradermally, intravitreally, subdurally, intraparenchymally, within the intracerebroventricular fluid or within the cerebrospinal fluid in the subarachnoid space.

[0025] In one embodiment, the implantable device is adapted to be implanted subdurally.DEFINITIONS

[0026] In the present invention, the following terms have the following meanings:

[0027] “About” : preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0028] “ARSA” or “Arylsulfatase A” refers to the enzyme that catalyzes the first step in the degradation pathway of 3-O-sulfogalactosylceramides (sulfatides). An example of human ARSA is provided with the reference UniProtKB - Pl 5289.

[0029] “C2C12 1ine”: refers to an immortalized mouse myoblast cell line. Said cell line can be found in the ATCC with the reference CRL-1772.

[0030] “Disease or condition affecting the nervous system”: refers to a disease or condition that affects the structure or function of the nervous system.

[0031] “Identity” or “identical”: when used in a relationship between the sequences of two or more amino acid sequences, or of two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity of related amino acid sequences or nucleicacid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5): 1073-82. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3) :403 - 10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may also be used to determine identity.

[0032] “Frataxin”: refers to a mitochondrial protein associated with cell iron metabolism. In human, the protein is encoded by the FXN gene. An example of human frataxin is provided with reference Uniprot - QI 6595.

[0033] “Friedreich ataxia”: refers to an autosomal recessive disease characterized by degeneration of the proprioceptive neurons in the dorsal root ganglia, the dentate nucleus, spinocerebellar tracts, posterior columns, and to a lesser extent the corticospinal tracts. Clinical manifestations include gait ataxia, pes cavus, speech impairment, lateral curvature of spine, rhythmic head tremor, kyphoscoliosis, congestive heart failure (secondary to a cardiomyopathy), and lower extremity weakness.

[0034] “Lysosomal storage diseases” or “LSD”: refers to inherited metabolic disorders characterized by defects in lysosomal function and resulting in intracellular accumulation of unmetabolized substrates.

[0035] “Mammal”: refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.

[0036] “Metachromatic leukodystrophy”: refers to a disease caused by an inherited deficiency of arylsulfatase A. Three clinical forms of MLD have been described, based on the age of symptom onset: late infantile, juvenile and adult forms.

[0037] “Mucopolysaccharidosis”: refers to a group of inherited metabolic diseases caused by the absence or malfunctioning of enzymes degrading glycosaminoglycans.

[0038] “N-acetyl-alpha-glucosaminidase” or “NAGLU”: refers to an enzyme which catalyses the hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues in N- acetyl-a-D-glucosaminides. An example of human NAGLU is provided with the reference UniProtKB - P54802.

[0039] “N-sulfoglucosamine sulfohydrolase” or SGSH refers to the enzyme sulfamidase, one of several enzymes involved in the lysosomal degradation of heparan sulfate. Mutations in this gene are associated with the lysosomal storage disease mucopolysaccharidosis IIIA, also known as Sanfilippo syndrome. An example of human SGSH is provided with the reference UniProtKB P51688.

[0040] “Nervous system”: refers to the organized network of nerve tissue in the body. In vertebrates, it includes the central nervous system (CNS) (i.e. the brain and spinal cord) and the peripheral nervous system (z.e. dorsal roots ganglia and nerves that extend from the spinal cord to the rest of the body).

[0041] “Primate”: refers to any member of the biological order Primates, the group that contains all the species commonly related to the lemurs, monkeys, and apes, with the latter category including humans.

[0042] “Progeny derived from primary human myoblast cells” refers to cells obtained by differentiation (e.g., by in vivo differentiation or in vitro differentiation) of primary human myoblast cells. An example of a progeny derived from primary human myoblast cells is the cell line deposited to CCOS under the accession number 1902.

[0043] “Sphingolipidosis”: refers to a group of inherited metabolic disorders characterized by the intralysosomal accumulation of sphingolipids primarily in the central nervous system and to a variable degree in the visceral organs. They are classified by the enzyme defect in the degradation pathway and the substrate accumulation.

[0044] “Subject”: as used herein, refers to a mammal, preferably a human. In one embodiment, a subject may be a patient, z.e., a warm-blooded animal, more preferably a human, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease.

[0045] “Therapeutically effective amount”: refers to the level or amount of genetically engineered cells loaded in the implantable device that is aimed at, without causing significant negative or adverse side effects to the target, i) delaying or preventing the onset of a disease, disorder, or condition; ii) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease or condition; iii) bringing about ameliorations of the symptoms of the disease or condition; iv) reducing the severity of the disease or condition; v) or curing the disease or condition. A therapeutically effective amount may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively or additionally, a therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.

[0046] “Treating” or “treatment”: refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject is successfully "treated" for a disease or condition if, after receiving the device according to the present invention, the subject showsobservable and / or measurable reduction in or absence of pathogenic cells and / or abnormalities associated with the specific disease or condition; relief to some extent of one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality; and / or improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0047] “Variant”: refers to functional conservative variants, i.e. variants in which one or more given amino acid residue(s) has / have been modified without altering the overall conformation and function of the proteins of interest, including, but not limited to, the replacement of one amino acid by another with similar properties (such as, for example, polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity and others).DETAILED DESCRIPTION

[0048] The present invention relates to a genetically engineered cell secreting a protein of interest for use in treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cell is encapsulated in an implantable device and wherein the disease or condition affecting the nervous system is either: i) a storage disease, such as a lysosomal storage disease, and the protein of interest is the wild-type version of the mutated protein causing the storage disease, or ii) the disease or condition is Friedreich ataxia, and the protein of interest is frataxin.

[0049] In one embodiment, the disease or condition affecting the nervous system is a storage disease, such as, for example, a lysosomal storage disease.

[0050] In one embodiment, the disease or condition affecting the nervous system is a storage disease selected from the group comprising or consisting of sphingolipidosis, and mucopolysaccharidosis (MPS).

[0051] In one embodiment, the disease or condition affecting the nervous system is a sphingolipidosis.

[0052] In one embodiment, the disease or condition affecting the nervous system is metachromatic leukodystrophy (MLD).

[0053] In one embodiment, the disease or condition affecting the nervous system is MLD. In one embodiment, the MLD is selected from the group consisting of the late infantile form, the juvenile form, and the adult form.

[0054] In one embodiment, the disease or condition affecting the nervous system is a MPS.

[0055] In one embodiment, the disease or condition affecting the nervous system is MPS type III (San Filippo syndrome).

[0056] In one embodiment, the disease or condition affecting the nervous system is a MPS type III. As used herein, MPS type III includes 4 sub-types: MPS type III A, MPS type III B, MPS type III C and MPS type III D. In one embodiment, the disease or condition affecting the nervous system is a MPS type III A. In one embodiment, the disease or condition affecting the nervous system is a MPS type III B.

[0057] In one embodiment, the disease or condition affecting the nervous system is a degenerative neuromuscular disease.

[0058] In one embodiment, the disease or condition affecting the nervous system is Friedreich ataxia.

[0059] In one embodiment, the disease or condition affecting the nervous system is a storage disease, such as a lysosomal storage disease, and the protein of interest is the wildtype version of the mutated protein causing the storage disease.

[0060] In one preferred embodiment, the disease or condition affecting the nervous system is to be treated in a human and the protein of interest is a human protein.

[0061] In one embodiment, the disease or condition affecting the nervous system is a sphingolipidosis and the protein of interest is an enzyme involved in the degradation of sphingolipids.

[0062] In one embodiment, the disease or condition affecting the nervous system is MLD, and the protein of interest is arylsulfatase A (ARSA).

[0063] In one embodiment, the disease or condition affecting the nervous system is the late infantile form of MLD, and the protein of interest is arylsulfatase A (ARSA). In one embodiment, the disease or condition affecting the nervous system is the juvenile form of MLD, and the protein of interest is arylsulfatase A (ARSA). In one embodiment, the disease or condition affecting the nervous system is the adult form of MLD, and the protein of interest is arylsulfatase A (ARSA).

[0064] In one embodiment, the protein of interest is human ARSA or a variant thereof. Example of a human ARSA is provided hereinabove.

[0065] In one embodiment, the protein of interest comprises or consists of the sequence SEQ ID NO: 1, or a variant sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 1.SEQ ID NO: 1MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGDLGCYGHPSSTTPNLDQLAA GGLRFTDFYVPVSLCTPSRAALLTGRLPVRMGMYPGVLVPSSRGGLPLEEVTV AEVLAARGYLTGMAGKWHLGVGPEGAFLPPHQGFHRFLGIPYSHDQGPCQNL TCFPPATPCDGGCDQGLVPIPLLANLSVEAQPPWLPGLEARYMAFAHDLMADA QRQDRPFFLYYASHHTHYPQFSGQSFAERSGRGPFGDSLMELDAAVGTLMTAI GDLGLLEETLVIFTADNGPETMRMSRGGCSGLLRCGKGTTYEGGVREPALAFWPGHIAPGVTHELASSLDLLPTLAALAGAPLPNVTLDGFDLSPLLLGTGKSPRQSL FFYPSYPDEVRGVFAVRTGKYKAHFFTQGSAHSDTTADPACHASSSLTAHEPPL LYDLSKDPGENYNLLGGVAGATPEVLQALKQLQLLKAQLDAAVTFGPSQVAR GEDPALQICCHPGCTPRPACCHCPDPHA

[0066] In one embodiment, the disease or condition is a mucopolysaccharidosis and the protein of interest is an enzyme involved in the degradation of glycosaminoglycans.

[0067] In one embodiment, the disease or condition is a mucopolysaccharidosis type III, preferably MPS type III A, and the protein of interest is SGSH.

[0068] In one embodiment, the protein of interest is human SGSH or a variant thereof. Example of a human SGSH is provided hereinabove.SEQ ID NO: 16MSCPVPACCALLLVLGLCRARPRNALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGVLNDTL VIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTI LDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVTMSYPMR SVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVC APDGVLEEKLSPQCQPLHNEL

[0069] In one embodiment, the disease or condition is a mucopolysaccharidosis type III, preferably MPS type III B, and the protein of interest is N-acetyl-alpha-glucosaminidase (NAGLU).

[0070] In one embodiment, the protein of interest is human NAGLU or a variant thereof. Example of a human NAGLU is provided hereinabove.

[0071] In one embodiment, the protein of interest comprises or consists of the sequence SEQ ID NO: 3, or a variant sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 3.SEQ ID NO: 3MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSVSVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCHVAWSGSQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARWEREI DWMALNGINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGN LHTWDGPLPPSWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQ VNVTKMGSWGHFNCSYSCSFLLAPEDPIFPIIGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATTAVYEAMTAVDTEAVWLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAESQPVYTRTASFQGQPFIWCMLHNFGGNHGLFGAL EAVNGGPEAARLFPNSTMVGTGMAPEGISQNEVVYSLMAELGWRKDPVPDLA AWVTSFAARRYGVSHPDAGAAWRLLLRSVYNCSGEACRGHNRSPLVRRPSLQ MNTSIWYNRSDVFEAWRLLLTSAPSLATSPAFRYDLLDLTRQAVQELVSLYYE EARS AYL SKELASLLRAGGVLAYELLPALDEVLASDSRFLLGSWLEQARAAAV SEAEADFYEQNSRYQLTLWGPEGNILDYANKQLAGLVANYYTPRWRLFLEAL VDSVAQGIPFQQHQFDKNVFQLEQAFVLSKQRYPSQPRGDTVDLAKKIFLKYY PRWVAGSW

[0072] As used herein, a protein is said to have a therapeutic effect on a disease or condition if the subject treated with the protein shows observable and / or measurable reduction in or absence of pathogenic cells and / or abnormalities associated with the specific disease or condition; relief to some extent of one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality; and / or improvement in quality of life issues.

[0073] In one preferred embodiment, the disease or condition affecting the nervous system is to be treated in a human and the protein of interest is a human protein.

[0074] In one embodiment, the disease or condition affecting the nervous system is Friedreich ataxia, and the protein of interest is frataxin.

[0075] In one embodiment, the protein of interest is human frataxin or a variant thereof. Example of human frataxins, including premature and mature forms, are provided hereinabove.

[0076] In one embodiment, the protein of interest is human frataxin in the premature form, or a variant thereof. In one embodiment, the protein of interest comprises or consists of the sequence SEQ ID NO: 7, or a variant sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 7.SEQ ID NO: 7MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA

[0077] In one embodiment, the protein of interest is human frataxin in the mature form, or a variant thereof. In one embodiment, the protein of interest comprises or consists of the sequence SEQ ID NO: 8, or a variant sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 8.SEQ ID NO: 8SGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTV KLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELL AAELTKALKTKLDLSSLAYSGKDA

[0078] It will be understood that the protein of interest as described herein can be modified to enhance its secretion, its recapture and / or its addressing to the cellular compartment enabling its function.

[0079] In one embodiment, the protein of interest is fused with one or several peptide(s) promoting protein secretion, protein recapture and / or protein addressing. In one embodiment, the protein of interest is fused with a peptide promoting protein recapture. In one embodiment, the protein of interest is fused with a peptide promoting its addressing to the cellular compartment enabling its function.

[0080] It will be understood that said peptides may be derived from a protein or engineered.

[0081] In one embodiment, the one or several peptide(s) promoting protein secretion, protein recapture and / or protein addressing is selected from the group comprising or consisting of a protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep-2 peptide (Ang-2), receptor-binding domains of human apolipoprotein B (ApoB) and ApoE, including ApoE-I and ApoE-II, a native secreted alkaline phosphatase (SEAP) secretion peptide, signal peptides derived from interleukins, and variants thereof.

[0082] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is a protein transduction domain of the human immunodeficiency virus TAT protein (Tat) or a variant thereof. Thus, in one embodiment, the protein of interest is one of the proteins described hereinabove, preferably ARSA, fused with a protein transduction domain of the human immunodeficiency virus TAT protein (Tat) or a variant thereof.

[0083] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing comprises or consists of the sequence SEQ ID NO: 9 or a variant thereof sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 9.

[0084] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is an Angiopep-2 peptide (Ang-2) or a variant thereof. Thus, in one embodiment, the protein of interest is one of the proteins described hereinabove, preferably ARSA, fused with an Angiopep-2 peptide (Ang-2) or a variant thereof.

[0085] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing comprises or consists of the sequence SEQ ID NO: 10 or a variant thereof sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 10.

[0086] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is a receptor-binding domain of human apolipoprotein B (ApoB) or a variant thereof. Thus, in one embodiment, the protein of interest is one of the proteins described hereinabove, preferably ARSA, fused with a receptor-binding domain of human apolipoprotein B (ApoB) or a variant thereof.

[0087] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing comprises or consists of the sequence SEQ ID NO: 11 or a variant thereof sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 11.

[0088] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is a receptor-binding domain of human apolipoprotein E (ApoE) or a variant thereof.

[0089] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is a receptor-binding domain of ApoE I or a variant thereof. Thus, in one embodiment, the protein of interest is one of the proteins described hereinabove, preferably ARSA, fused with a receptor-binding domain of ApoE I or a variant thereof.

[0090] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing comprises or consists of the sequence SEQ ID NO: 12 or a variant thereof sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 12.

[0091] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is a receptor-binding domain of ApoE II or a variant thereof. Thus, in one embodiment, the protein of interest is one of the proteins described hereinabove, preferably ARSA, fused with a receptor-binding domain of ApoE II or a variant thereof.

[0092] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing comprises or consists of the sequence SEQ ID NO: 13 or a variant thereof sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 13.

[0093] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing is a native secreted alkaline phosphatase (SEAP) secretion peptide. Thus, in one embodiment, the protein of interest is one of the proteins described hereinabove, preferably ARSA, fused with a native secreted alkaline phosphatase (SEAP) secretion peptide or a variant thereof.

[0094] In one embodiment, the peptide promoting protein secretion, protein recapture and / or protein addressing comprises or consists of the sequence SEQ ID NO: 14 or avariant thereof sharing at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more identity with SEQ ID NO: 14.SEQ ID NO: 9 = YGRKKRRQRRRSEQ ID NO: 10 = TFFYGGSRGKRNNFKTEEYSEQ ID NO: 11 = SVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGSSEQ ID NO: 12 = TEELRVRLASHLRKLRKRLLRDASEQ ID NO: 13 = LRI<LRI<RLLLRI<LRI<RLLSEQ ID NO: 14 = MLLLLLLLGLRLQLSLG

[0095] In one embodiment, the protein of interest is fused with a tag. An example of a tag is the HA-tag derived from the human influenza hemagglutinin (HA) and corresponding to amino acids 98-106.

[0096] In one embodiment, the subject is affected or diagnosed with one of the diseases or conditions described hereinabove, and is presymptomatic (i.e. the subject does not present symptoms of the disease or condition yet).

[0097] In one embodiment, the subject is affected or diagnosed with one of the diseases or conditions described hereinabove, and is symptomatic (i.e. the subject presents at least one symptom of the disease or condition).

[0098] In one embodiment, the subject is affected or diagnosed with MLD, and is presymptomatic.

[0099] In one embodiment, the subject is affected or diagnosed with MLD, and is symptomatic. In one embodiment, said subject presents at least one of the symptoms described hereinbelow. In one embodiment, said subject present at least one of the abnormalities described hereinbelow.

[0100] Examples of symptoms of MLD include, without limitation, motor impairment, coordination loss, and cognitive impairment.

[0101] Examples of abnormalities associated with MLD include, without limitation, sulfatide accumulation, myelin abnormalities on MRI, neuronal loss, neuroinflammation, and abnormal nerve conduction velocity.

[0102] In one embodiment, the symptomatic subject presents with sulfatide accumulation and neuroinflammation.

[0103] In one embodiment, the subject is affected or diagnosed with MLD and is early symptomatic. As used herein, said subject is early symptomatic if said subject presents with signs of clinical manifestations but does not present diffuse areas of demyelination nor clinical abnormalities such as behavior impairment.

[0104] In one embodiment, said subject is affected or diagnosed with MLD and is late symptomatic. As used herein, said subject is late symptomatic if said subject presents with signs of clinical manifestations and diffuse areas of demyelination and severe sulfatide accumulation, and neuroinflammation.

[0105] In one embodiment, the subject is affected or diagnosed with MLD and has received or is to be received a transplantation of hematopoietic stem cells secreting the protein of interest, preferably ARSA, in the brain.

[0106] In one embodiment, the hematopoietic stem cells were transduced with a lentivirus (i.e. hematopoietic lentiviral corrected hematopoietic stem cells) to secret the protein of interest, preferably ARSA, in the brain.

[0107] In one embodiment, the subject is affected or diagnosed with MLD and has received or is to be received Libmeldy© medicine.

[0108] In one embodiment, the subject is affected or diagnosed with MLD and has received or is to be received a vector, such as, for example, a viral vector, encoding the protein of interest, preferably ARSA, in the brain.

[0109] In one embodiment, the subject is affected or diagnosed with MLD and has received or is to be received an IT-ERT with the protein of interest, preferably ARSA.

[0110] In one embodiment, the subject is affected or diagnosed with MLD and has received or is to be received a weekly chronic injection of the protein of interest, preferably ARSA.

[0111] In one embodiment, the subject is receiving, has to be received or will receive an anti-inflammatory compound.

[0112] Examples of anti-inflammatory compounds include, without limitation, leriglitazone and dendrimers.

[0113] According to the invention, the genetically engineered cell is encapsulated in a device, i.e. the cell is confined in the device.

[0114] In one embodiment, the cell to be used is genetically engineered to secrete the protein of interest, i.e. the cell to be used does not naturally secrete the protein of interest.

[0115] It will be understood that the genetically engineered cell to be used may be adapted depending on the protein of interest to be secreted. It will be understood that the genetically engineered cell to be used may also be adapted depending on the implant location.

[0116] In one embodiment, the genetically engineered cell is a human cell.

[0117] In one embodiment, the genetically engineered cell is an immortalized human myoblast cell, or a progeny thereof derived from primary human myoblast cells. In one embodiment, the genetically engineered cell is a cell from the immortalized human myoblast cell line deposited with CCOS under accession number 1902, or a progeny thereof. This cell line was deposited with CCOS under accession number 1902 by MaxiVAX SA (which changed its name to Release Therapeutics SA) and UNITEC - Universite de Geneve.

[0118] In one embodiment, the genetically engineered cell is a cell from the immortalized human myoblast cell line deposited with CCOS under accession number 1902, or a progeny thereof, secreting ARSA.

[0119] In one embodiment, the genetically engineered cell is transformed with an expression vector enabling the secretion of the protein of interest.

[0120] Examples of vectors include, without limitation, plasmids, viral vectors, non- viral gene delivery system, and vectors harboring eukaryotic transposable elements.

[0121] In one embodiment, the genetically engineered cell is transformed with a viral vector enabling the secretion of the protein of interest.

[0122] Examples of viral vectors include adenoviruses, retroviruses including lentiviruses, poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses.

[0123] In one embodiment, the expression vector is a lentivirus enabling the secretion of the protein of interest.

[0124] In one embodiment, the cell is genetically engineered with a transposable element enabling the secretion of the protein of interest.

[0125] In one embodiment, the device is an implantable device, i.e. a device adapted to be placed in a body either permanently or temporarily.

[0126] In one embodiment, the device is placed permanently in the body, i.e. the device is not to be removed from the body once implanted.

[0127] In one embodiment, the device is placed temporarily in the body, i.e. the device is to be removed from the body once implanted. In one embodiment, the device is to be removed about 4 months, 6 months, 1 year, 2 years, 5 years, 10 years or more after implantation. It will be understood that once the device is removed, a new device replacing the old one can be placed in the body.

[0128] In one embodiment, the device is made of biocompatible materials.

[0129] In one embodiment, the device is semi-porous. According to the invention, a semi-porous device allows the secretion of the protein of interest out of the device and protects the engineered cells against the immunological response of circulating cells.Thus, as used herein, a semi-porous device is a device having pores size enabling the crossing of proteins but impermeable to cells.

[0130] It will be understood that the shape and the volume of the device may vary according to the subject to be implanted and the place of implantation.

[0131] In one embodiment, the device is an implantable capsule.

[0132] In one embodiment, the implantable capsule comprises a cell receiving portion and a cell receiving chamber separated by a semi-porous membrane.

[0133] In one embodiment, when the capsule is to be implanted temporarily, the capsule further comprises an extractor portion. This extractor portion may have different shapes and configurations, and aims at allowing a surgical device to grip on the implant and to pull it out of the subject’s tissue.

[0134] In one embodiment, when the capsule is to be implanted subdurally, the capsule does not comprise an extractor portion, to avoid damaging the parenchyma tissues.

[0135] Examples of implantable capsules are provided in Figures 9 to 13.

[0136] By reference to Figures 9 to 13, an example of implantable capsule (1) as described herein, comprises a cell receiving portion (2) and an extractor portion (3) connected together by a coupling (4). The cell receiving portion (2) comprises a substantially cylindrical outer shape with a diameter that may typically be in the range of 0.5 to 3 millimeters and a length that may be typically in the range of 5 to 20 millimeters for instance around 10 millimeters. The ratio L / D of the length L to the diameter D is preferably in a range from 5 to 20 preferably in a range from 5 to 15.

[0137] The cell receiving portion (2) comprises a porous membrane (5) that is configured for allowing the proteins of interest produced by the cells (24) contained in the capsule to pass through the membrane to the surrounding tissue and to allow fluids and electrolytes and nutrients for the cells (24) to pass into the capsule through the membrane from the surrounding tissue.

[0138] The porosity and the type of membrane may thus depend on the specific application and type of cells that are contained within the capsule. In an example, the membrane is for instance in a form of a polyether-sulfone (PES) membrane having a porosity around 0.65pm configured to allow the target proteins to pass though the membrane. An example of a membrane may be used in the present invention is detailed hereafter.

[0139] An exemplary embodiment of a membrane comprises polyethersulfone based on its biocompatible chemical composition, the structure properties as well as the inherent membrane performance, such as the superior flow rates, the downstream cleanliness and the low protein binding affinity. It can be extruded in different shapes and in small diameter tubing.

[0140] The cell receiving portion (2) can have the form of a flat sheet as described in Lathuillere et al., 2014, Biomaterials 35 780-790 or WO2014 / 173441 or hollow fiber such as described in Lathuillere et al., 2015, Int. J. Mol. Sci., 16, 10578-10600, depending on the secreted protein and the site of implantation of the device.

[0141] In one embodiment, the porous membrane (5) surrounds a cell receiving chamber (13) and a membrane support (6) within the cell receiving chamber (13). The membrane support serves to mechanically support the porous membrane to maintain the stability of the volume in the cell receiving chamber (13) and to prevent rupture of the membrane. In the illustrated embodiment, the membrane support is in a form of a coil, in particular a stainless-steel coil that is per se known for instance as described in WO2017 / 0645701. The membrane support 6 also serves to anchor the extractor portion (3) via the coupling (4).

[0142] In the illustrated embodiment, the coupling (4) comprises a connector (10) having a portion (10a) that is inserted into the membrane support (6), in particular within the cylinder surrounded by the stainless-steel coil in the present example. The diameter of the connector insert portion (10a) may be configured to engage in a tight fit in an extractor end of the coil for a stable connection therebetween. The coupling (4) further comprises a fixing portion (10b) engaging an anchor (8) of the extractor portion (3), whereby in thepresently illustrated embodiment the anchor (8) is in the form of a tube, preferably a polyurethane (PU) tube fitted over the second end (10b) of the connector (10). An adhesive (18a) may be deposited on the connector prior to insertion of the exactor end (12b) of the cell receiving portion, respectively coupling end (8a) of the anchor (8) on the connector (10). The adhesive may advantageously be in the form of a light curable adhesive for instance of the type of photocurable urethane methacrylate (such as Dymax 1187 M SV).

[0143] In one embodiment, the extractor portion (3) serves to provide a means to pull out the implant from the subject’s tissue at the end of its use. In the illustrated embodiment, the extractor portion further comprises a retrieval thread (9) comprising an anchor portion (15) fixed to the anchor tube (8) and a thread portion (16) extending beyond the anchor tube configured for allowing a surgical device to catch the thread to pull out the implantable capsule. In the illustrated embodiment, the retrieval thread (9) is made of a biocompatible yarn or thread, for instance of the type of Polypropylene (such as the Prolene ™ suture).

[0144] In one embodiment, a length of the thread extends within the hollow anchor tube 8 and comprises knots (15a), the anchor portion (15) being held within the tube by an adhesive, for instance a light curable adhesive as described above, the knots increasing the strength of the attachment of the retrieval thread to the anchor tube. The retrieval thread is thus supple and very fine to reduce discomfort of the subject and allow easy removal of the implant.

[0145] In one embodiment, the retrieval thread may be directly fixed to the connector (10), or be integrally formed with the connector (10), without the presence of the anchor tube. In such a variant, the connector may for instance comprise an orifice allowing a thread to feed through the orifice and allow for pulling the implanted capsule out of the subject’s tissue. The polyurethane tube, or any other material with the adequate mechanical and biological properties, advantageously provides a structure which supports the coupling of the retrieval thread. It may also be used as the support for the tweezers during manipulations, whether it is during the assembly or the implantation.

[0146] In one embodiment, the cell receiving portion (2) further comprises a cell support matrix (7) inserted within the cell receiving chamber. In one embodiment, the cell support matrix can be composed of biocompatible materials, basement membrane matrix or hydrogel. In a preferred embodiment, the cell support matrix (7) comprises one or more yams (14) of a biocompatible material, preferably a plurality of yams extending longitudinally within the cell receiving chamber (13). The yams extend, in a preferred embodiment, from a position at or proximate the extractor end (12b) to a position at or proximate a cell loading end (12a) of the membrane (5). The yarns preferably extend over the entire length or a large portion of the length of the cell receiving chamber (13). In a preferred embodiment, the yarns may advantageously be made of polyester (PE) of clinical grade that is per se well-known and already approved for surgical implantation uses. Such polyester yams are typically used for sutures of tissue within a subject’s body. An example of polyester yarns that may be used in advantageous embodiments of the invention is for instance 44 / 27-PET-5540-FTT-SS (Textile Development Associates, Inc). The material is 40 denier 27 filaments yarn made of textured polyester.

[0147] It has been found that the cell support matrix (7) significantly improves the performance of the cells contained in the cell receiving chamber, increasing both activity in a release of proteins of interest and durability over time, in particular for adherent cells. Whereby it is being found that these cells (24) tend to align with the fibers of the yarn (14) thus improving the density and spacing of the cells optimized for release of proteins of interest and ingestion of nutriments. The yams also advantageously provide a large overall surface area for adherents of cells thereto.

[0148] In an exemplary embodiment, a cell receiving cavity (13) may receive therein for instance five to twenty yarns (14) arranged in parallel within the cavity extending substantially the whole length of the cavity. The yarns may be inserted within the cell receiving cavity (13) by pulling one end thereof through the cavity, the coupling (4) being mounted on the extractor end (12b) of the cell receiving portion (2) once the membrane support (6) and cell support matrix (14) have been mounted in the porous membrane (5).

[0149] It may be noted that the cell support matrix (7) may be preassembled to the membrane support (6), for instance by inserting them through the inside of the coil priorto the insertion of the preassembled coil and cell support matrix into the tubular porous membrane (5).

[0150] The cell support matrix (7), in particular in the form of yarns (14), thus has a very beneficial effect of optimizing the distribution of cells in an orderly manner within the cell receiving chamber, improving the secretion yield and rate for a given volume. Moreover, this configuration allows the length of the cell receiving portion to be easily modified by simply changing the cut length of the yarns to the corresponding length of the porous membrane tube and coil of the membrane support (6). Moreover, the use of well characterized biocompatible implantable polyester does not adversely impact the safety of the device.

[0151] It has also been observed that the presence of the cell support matrix (7) allows the freezing and thawing of cells contained without affecting the viability of the cells. The presence of the matrix (7) improves the freezing and thawing properties of the capsule which is particularly advantageous since it allows the capsules to be stored for extended periods of time in a frozen state, ready for use in the treatment of a subject when needed. In particular, it would appear that the improved distribution of cells, in particular adherent cells, along the yams contribute to maintain a high rate of viability during the freezing and thawing process.

[0152] The cells in a liquid media may be inserted into the cell receiving chamber (13) by means of a cell loading device (20) (only partially and schematically represented in the illustrations) comprising an outlet nozzle (22) that is inserted in a cell loading end (12a) of the porous membrane (5).

[0153] The implantable capsule (1) may be supplied in a preassembled arrangement with the cell loading device. In this embodiment, the nozzle (22) of the cell loading device may be attached to the cell loading end (12a) of the membrane, for instance by means of an adhesive (18c), for instance a light curable adhesive as already described hereinabove. The cell loading device may comprise a catheter tube to allow the cells in a liquid media to be injected through the catheter tube into the cell receiving chamber of the capsule, aircontained within the cell receiving capsule being pushed out through the porous membrane (5).

[0154] Thus, in one embodiment, the implantable capsule comprises a cell receiving portion (2) comprising a porous membrane surrounding a cell receiving chamber for receiving immortalized cells in a liquid media, and a cell support matrix comprising at least one yarn inserted within the cell receiving chamber configured for the arrangement of the immortalized cells within the cell receiving chamber.

[0155] In one embodiment, the implantable capsule as described hereinabove comprises one or several of the characteristics hereinbelow.

[0156] In one embodiment, in the implantable capsule as described hereinabove, said at least one yam consists of or comprises a polyester material.

[0157] In one embodiment, in the implantable capsule as described hereinabove, the cell support matrix 7 comprises a plurality of said yams.

[0158] In one embodiment, in the implantable capsule as described hereinabove, the plurality of yams is in a range of 5 to 20 yarns, preferably a range of 5 to 15 yarns, for instance around 10 yarns.

[0159] In one embodiment, in the implantable capsule as described hereinabove, the yams extend within the cell receiving chamber over substantially the whole length of the chamber or at least 80 per cent of the length of the cell receiving chamber.

[0160] In one embodiment, in the implantable capsule as described hereinabove, the cell receiving chamber (13) comprises polyester yams.

[0161] In one embodiment, in the implantable capsule as described hereinabove, the cell receiving portion further comprises a membrane support (6) mounted in the cell receiving chamber (13) configured for providing structural support to the porous membrane (9), the membrane support consisting of or comprising a coil made of a biocompatible material, for instance of stainless-steel coil.

[0162] In one embodiment, the implantable capsule as described hereinabove further comprises an extractor portion (3) coupled to an extractor end (12a) of the cell receiving portion configured for allowing a surgical tool to pull the implantable capsule out of an implantation site, the extractor portion comprising a retrieval thread (9).

[0163] In one embodiment, in the implantable capsule as described hereinabove, the retrieval thread (9) is made of a thread of polypropylene.

[0164] In one embodiment, in the implantable capsule as described hereinabove, the extractor portion comprises an anchor tube (8) having a cavity therein within which an anchor portion (15) of a retrieval thread (9) is inserted and bonded.

[0165] In one embodiment, in the implantable capsule as described hereinabove, the anchor tube (8) consists of or comprises a polyurethane material.

[0166] In one embodiment, in the implantable capsule as described hereinabove, the extractor portion (3) is coupled to the cell receiving portion (2) via a coupling (14) comprising a connector (10), a connector (10) comprising a portion (10a) inserted into an extractor end (12b) of the porous membrane (5) and second portion (10b) inserted in a coupling end (8a) of the anchor tube (8).

[0167] In one embodiment, in the implantable capsule as described hereinabove, the connector (10) is bonded to the anchor tube (8) and the cell receiving portion (2) by an adhesive (18b), in particular a light curable adhesive, for instance of the type of photocurable urethane methacrylate.

[0168] In one embodiment, an outer diameter of the implantable capsule as described hereinabove is in a range of 0.5 to 3 millimeters preferably in a range of 0.8 to 1.5 millimeters and has a length in a range of 5 to 25 millimeters preferably in a range of 8 to 20 millimeters.

[0169] In one embodiment, a length to diameter ratio of the implantable capsule as described hereinabove is in a range of 5 to 20.

[0170] In one embodiment, the implantable capsule is coated with an antifibrotic agent to limit the formation of fibrosis, preferably GW2580.

[0171] Exemplary implantable capsules are provided in the patent application WO202 1028502, which is incorporated herein by reference.

[0172] It will be understood that the number of cells to be loaded in the device can vary depending on the volume of the device, and in particular, the volume of the cell-receiving chamber, and the growth rate of the cells.

[0173] In one embodiment, the implantable device comprises a therapeutically effective amount of cells, i.e. an amount of cells enabling secretion of the protein of interest in an amount sufficient to have a therapeutical effect in the subject.

[0174] In one embodiment, the implantable device comprises from about 100 000 to about 2 million of cells, preferably from about 150 000 to about 1.5 million of cells, more preferably from about 250 000 to about 1 million of cells.

[0175] Those skilled in the art will recognize that the exact cell number in the implantable device can depend both upon the growth rate of the cell / cell line encapsulated and / or the volume of the implantable device (in particular, of the cell receiving portion).

[0176] In one embodiment, the cells are loaded in the device in a cell growth medium suitable for the type of cells such as Ham’s F12 or DMEM supplemented with growth factors or fetal bovine serum. Further, for cells that will be frozen (see above), a freezing medium / cryopreservant may also be added to the cell growth medium, such as glycerol.

[0177] In one embodiment, the implantable device is adapted to be implanted within the body of the subject. In one embodiment, the step of implanting the device in the body of the subject is not part of the invention.

[0178] In one embodiment, the implantable device is adapted to be implanted subcutaneously, intramuscularly, intradermally, intravitreally, subdurally, intraparenchymally, or within the intracerebroventricular fluid.

[0179] In one embodiment, the implantable device is adapted to be implanted subcutaneously, intramuscularly, intradermally, intravitreally, subdurally, intraparenchymally, within the intracerebroventricular fluid or within the cerebrospinal fluid in the subarachnoid space.

[0180] In one embodiment, the implantable device is adapted to be implanted subcutaneously (i.e. in the subcutaneous tissue, located below the dermis and epidermis).

[0181] In one embodiment, the implantable device is adapted to be implanted intramuscularly (z.e. into a muscle).

[0182] In one embodiment, the implantable device is adapted to be implanted intradermally (z.e. into the dermis, which is located between the epidermis and the hypodermis).

[0183] In one embodiment, the implantable device is adapted to be implanted intravitreally (z.e. into the vitreous humor of the eye).

[0184] In one embodiment, the implantable device is adapted to be implanted subdurally (i.e. into the subdural space).

[0185] In one embodiment, the implantable device is adapted to be implanted intraparenchymally (i.e. into the parenchyma).

[0186] In one embodiment, the implantable device is adapted to be implanted within the intracerebroventricular fluid.

[0187] In one embodiment, the implantable device is adapted to be implanted within the cerebrospinal fluid in the subarachnoid space.

[0188] It will be understood that several implantable devices may be administered to the same subject. Thus, the subject may comprise several implantable devices in several body parts.

[0189] Another object of the present invention is a method for treating a disease or condition affecting the nervous system in a subject in need thereof, the methodcomprising the step of implanting a device comprising encapsulated genetically engineered cells in the subject.

[0190] The disease or condition affecting the nervous system, the device, the genetically engineered cells and the subject are as described hereinabove.

[0191] The number of cells loaded in the device and the implantation pathways are as described hereinabove.

[0192] In one embodiment, the implantable device is to be implanted subdurally. In one embodiment, the implantable device is to be implanted subdurally in one or more locations, such as one or more locations in the central nervous system and / or in the peripheric nervous system.

[0193] In a particular embodiment, the implantable device is to be implanted subdurally in one or more locations selected from cerebral hemispheres, cistema magna, and dorsal root ganglia.

[0194] In one embodiment, the method of the present invention is for treating MLD in the subject.

[0195] In one embodiment, the step of implanting the device is carried out in a subject suffering or diagnosed with MLD and being presymptomatic. In another embodiment, the step of implanting the device is carried out in a subject suffering or diagnosed with MLD and being symptomatic.

[0196] In one embodiment, the method of the present invention further comprises a step of transplanting hematopoietic stem cells, preferably lentiviral corrected hematopoietic stem cells, secreting the protein of interest, preferably ARSA, in the nervous system of the subject to be treated.

[0197] Thus, in one embodiment, the method of the present invention comprises: i) a step of implanting a device comprising encapsulated genetically engineered cells as described hereinabove, andii) a step of transplanting hematopoietic stem cells, preferably lentiviral corrected hematopoietic stem cells, secreting the protein of interest, preferably ARSA, in the brain.

[0198] In one embodiment, the step (i) is implemented before the step (ii). In another embodiment, the step (i) is implemented after the step (ii). In another embodiment, the step (i) and (ii) are implemented at the same time.

[0199] In one embodiment, the method of the present invention further comprises a step of administering a vector, such as, for example, a viral vector, secreting the protein of interest, preferably ARSA, in the nervous system of the subject to be treated.

[0200] Thus, in one embodiment, the method of the present invention comprises: i) a step of implanting a device comprising encapsulated genetically engineered cells as described hereinabove, and ii) a step of administering a vector, such as, for example, a viral vector, secreting the protein of interest, preferably ARSA, in the brain of the subject.

[0201] In one embodiment, the step (i) is implemented before the step (ii). In another embodiment, the step (i) is implemented after the step (ii). In another embodiment, the step (i) and (ii) are implemented at the same time.

[0202] In one embodiment, the method of the present invention further comprises a step of administering an anti-inflammatory compound to the subject.

[0203] Thus, in one embodiment, the method of the present invention comprises: i) a step of implanting a device comprising encapsulated genetically engineered cells as described hereinabove, and ii) a step of administering an anti-inflammatory compound to the subject.

[0204] Examples of anti-inflammatory compounds are provided hereinabove.

[0205] In one embodiment, the step (i) is implemented before the step (ii). In another embodiment, the step (i) is implemented after the step (ii). In another embodiment, the step (i) and (ii) are implemented at the same time.

[0206] The present invention also relates to the use of a genetically engineered cell secreting a protein of interest for treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cell is encapsulated in an implantable device.

[0207] The present invention also relates to the use of a genetically engineered cell secreting a protein of interest in the manufacture of a medicament for treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cell is encapsulated in an implantable device.

[0208] The disease or condition affecting the nervous system, the device, the genetically engineered cells and the subject are as described hereinabove.

[0209] The number of cells loaded in the device and the implantation pathways are as described hereinabove.

[0210] Another object of the present invention is providing a method for treating a disease or condition affecting the nervous system in a subject in need thereof, the method comprising: i. providing an implantable device comprising genetically engineered cells secreting a protein of interest, wherein said cells are encapsulated in said implantable device; and ii. implanting said device subdurally in one or more locations selected from cerebral hemispheres, cisterna magna, and dorsal root ganglia from said subject; wherein: iii. the disease or condition affecting the nervous system is either: i) a storage disease, such as a lysosomal storage disease, and the protein of interest is the wild-type version of the mutated protein causing the storage disease, or ii) Friedreich ataxia, and the protein of interest is frataxin; and iv. wherein the implantable device comprises from about 100,000 to about 2 million of said genetically engineered cells.

[0211] It will be understood that the use of an implantable device comprising encapsulated genetically engineered cells for treating diseases or conditions affecting the nervous system may present several advantages, as described hereinbelow.

[0212] The use of an implantable device according to the invention may allow a quick secretion of proteins of interest in the brain (in particular, a secretion in few weeks) but also in the whole nervous system, which makes this cell-based therapy compatible with both slowly and rapidly progressing diseases or conditions and both presymptomatic and symptomatic subjects.

[0213] The use of an implantable device according to the invention may allow a high yield of release of proteins of interest, which makes this cell-based therapy compatible with both slowly and rapidly progressing diseases or conditions and both presymptomatic and symptomatic subjects.

[0214] The use of an implantable device, in particular adapted for subdural implantation, according to the invention may allow the release in the brain of proteins that cannot cross the blood brain barrier, which makes this cell-based therapy compatible with proteins of interest of any size.

[0215] The use of an implantable device, in particular adapted for subdural implantation, according to the invention may allow the release of proteins in sufficient amount in the brain and spinal cord to have a therapeutic effect, in particular in symptomatic subjects.BRIEF DESCRIPTION OF THE DRAWINGS

[0216] Figure 1 is a combination of histograms showing the validation of genetically engineered cells: C2C12-LV-hARSA-HA (called hARSA) and C2C12-LV-ApoE- hARSA-HA (called ApoE-hARSA). Figure 1A: Copy number of hARSA vector in nontransduced C2C12 cells (NT; n=5) and C2C12 transduced with hARSA (n=5) or ApoE- hARSA (n=3). VGC for vector genome copy number per 2n genome. Figure IB: ARSA activity on pellet or supernatant of non-transduced C2C12 (NT) and C2C12 transduced with hARSA or ApoE-hARSA. Figure 1C: hARSA expression on supernatant of non-transduced C2C12 (NT) and C2C12 transduced with hARSA or ApoE-hARSA. A significant expression of an active ARSA enzyme was detected in the two cell lines. Data are represented as mean + / - SEM.

[0217] Figure 2 is a combination of histograms showing the validation of hARSA secretion in genetically engineered cells: C2C12-LV-hARSA-HA (called hARSA) and C2C12-LV-ApoE-hARSA-HA (called ApoE-hARSA) before and after implantation in mice. Figure 2A: ARSA activity on cells or supernatant from non-transduced C2C12 (NT) and C2C12 transduced with hARSA or ApoE-hARSA before loading cells into devices that will subsequently be implanted in mice (n=l-2). Figure 2B: ARSA quantification on cells or fluid within device from non-transduced C2C12 (NT; n=2) and C2C12 transduced with hARSA (n=2-5) or ApoE-hARSA (n=3-6) after implantation in mice. Figure 2C: ARSA activity on cells or supernatant from non-transduced C2C12 (NT) and C2C12 transduced with hARSA or ApoE-hARSA after explantation in mice. Figure 2D: ARSA activity in several brain regions, spinal cord and kidney in 9-month- old control (n=5), untreated (n=5) and KO ARSA mice treated with device loaded with 250 000 or 1 million cells C2C12-hARSA (n=3-5) or ApoE-hARSA (n=5-6). Data are represented as mean + / - SEM. *p<0.05; **p<0.01; ****p<0.0001.

[0218] Figure 3 is combination of histograms showing the post-surgery score 3 days, 7 days or the day of necropsy (i.e. 3 months) in sham mice or implanted mice with nontransduced or transduced cells. The post-surgery score was low 7 days after implantation, indicating a good tolerance of device.

[0219] Figure 4 is a combination of histograms showing the biocompatibility of devices in implant groups. Figure 4A: Quantification of thickness between epidermis and muscle and the fibrotic capsule in sham and implanted groups. Figure 4B: Mean area of adipose tissue in sham and implanted groups. Figure 4C: Percentage of muscle fibers in degeneration in sham and implanted mice. Figure 4D: Quantification of neovascularization in fibrotic tissue of implanted mice after implantation. Figure 4E: Quantification of mast cell number by mm2in different groups. Figure 4F: Quantification of DAPI-stained sections. Groups show no significant difference. One-way ANOVA andTurkey’s test *p < 0.05; **p < 0.01; ***p < 0.0010.0001. * corresponds to p value comparing to SHAM.

[0220] Figure 5 is a combination of histograms showing the correction of sulfatide storage in brain and spinal cord of treated MLD mice, 3 months after treatment. Quantification of sulfatide storage per mm2in cerebral cortex (A), corpus callosum (B), fimbria (C), cerebellar white matter (D) and spinal cord (E) of WT, untreated (NT) and treated MLD mice. Data are represented as mean + / - SEM. One-way ANOVA and Turkey’s test *p < 0.05; **p < 0.01; ***p < 0.001 and0.0001. * corresponds to p value comparing to (1), $ correspond to p value comparing to (2), £ corresponds to p value comparing to (3), # corresponds to p value comparing to (4) and + corresponds to p value comparing to (5).

[0221] Figure 6 is a combination of histograms showing the sulfatide storage in sciatic nerve, gallbladder and kidney of treated MLD mice, 3 months after implantation. Quantification of sulfatide storage per mm2in sciatic nerve (A), percentage of positive area in gallbladder (B) and percentage of positive cells in kidney (C) of WT, untreated (NT) and treated MLD mice. Data are represented as mean + / - SEM. One-way ANOVA and Turkey’s test *p < 0.05; **p < 0.01; ***p < 0.001 and ****p< 0.0001. * corresponds to p value comparing to (1), $ correspond to p value comparing to (2)

[0222] Figure 7 is a histogram showing the correction of microgliosis in brain of treated MLD mice, 3 months after implantation. It shows the quantification of Ibal -positive cells per mm2in brain of WT, untreated (NT) and treated MLD mice. Data are represented as mean + / - SEM. *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001. grey stars correspond to p value comparing to (1), black stars correspond to p value comparing to (2).

[0223] Figure 8 is a histogram showing ARSA activity in a non-human primate after implantation of the cell-based device secreting ARSA, at the following time points: baseline, 1-week post-implantation, 1 -month post-implantation and 6 weeks postimplantation.

[0224] Figure 9 is a schematic illustration of an implantable capsule according to an embodiment of this invention.

[0225] Figure 10 is a cross-sectional view through line II-II of Figure 9.

[0226] Figure 11 is a cross-sectional view similar to Figure 10 with an internal matrix of the capsule removed.

[0227] Figure 12 is a detailed view of circle IV of Figure 11.

[0228] Figure 13 is a detailed view of circle V of Figure 10.

[0229] Figure 14 is a schematic illustration of the second experimental design, in NHP with subdural implantation. Black tubes are for blood sampling and white tubes for cerebrospinal fluid sampling.

[0230] Figure 15 is a histogram showing ARSA activity in CSF over in life follow up demonstrating increase of ARSA activity after subdural implantation.

[0231] Figure 16 is a combination of histograms showing ARSA activity in CNS (Figure 16A), in spinal cord and peripheral nervous system (Figure 16B), and in peripheral tissues (Figure 16C) at necropsy from non-human primates having received subdurally 3 capsules containing each one 1 million of ARSA or ApoE-ARSA secreting cells. Dotted line in Figures 16A and 16B corresponds to value in non-treated NHP. DRG: dorsal root ganglia.

[0232] Figure 17 is a schematic illustration of the third experimental design, for subcutaneous implantation in NHP. Black tubes are for blood sampling and white tubes for cerebrospinal fluid sampling.

[0233] Figure 18 is a combination of histograms showing ARSA activity in CNS (Figure 18A), and in spinal cord and peripheral nervous system (Figure 18B) at necropsy from non-human primates having received 5 for 3 months and 10 capsules for 2 months subcutaneously containing each one 1 million of ARSA or ApoE-ARSA secreting cells. Dotted line in Figures 18A and 18B corresponds to value in non-treated NHP. DRG: dorsal root ganglia.EXAMPLES

[0234] The present invention is further illustrated by the following examples.Example 1 : Cell-based therapy in miceMaterials and MethodsAnimal model

[0235] All animal studies were performed in accordance with local and national regulations and were reviewed and approved by the relevant institutional animal care and use committee. The experiments were carried out in accordance with the European Community Council directive (2010 / 63 / EU) for the care and use of laboratory animals. Our protocol was approved by the European community council directive (2010 / 63 / EU) (no. #21908).

[0236] Immunotol erant MLD mice (mARSA- / - hARSAC69S mice, named MLD mice) are tolerant to human ARSA and murine ARSA knock-out and were bred from heterozygous founders (Matzner et al. Mol. Med. 13, 471-479. 10.2119 / 2007-00063). Littermate mice (Tg ARSA+ / -) are used as wild-type for experiments. Mice were housed in a pathogen free animal facility in a temperature-controlled room and maintained on a 12-h light / dark cycle. Food and water were available ad libitum. Mice are genotyped by PCR as described previously (see Matzner supra).Genetically engineered cells

[0237] C2C12 cell line, an immortalized murine myoblast cell line, was transduced using two different lentiviral vectors (LV) constructions. The first construction was the LV- hARSA-HA. In the second construction, the ApoE-II-derived signal peptide was inserted into the hARSA-HA plasmid (ApoE-hARSA-HA). After validation of the construction by sequencing, a production of LV has been performed. The LV-hARSA-HA (titer 2.44.109 TU / ml) and LV- ApoE-hARSA-HA (titer 4.27 and 7.58.107TU / ml) were transduced at 50 MOI in C2C12 cell line. We have obtained polyclonal C2C12-LV-HA- hARSA and C2C12-LV-ApoE-HA-hARSA cell lines, later called hARSA and ApoE-hARSA respectively.

[0238] Cells were grown at 37°C in the presence of 5% CO2 in DMEM (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS, Thermo Fischer Scientific) and 0.2% MycoZap antibiotics (Lonza). The same cell passage was used to analyze / study comparable cells between each implantation. Thus, large batches of 2 million cells / vial were stored in the working biobank at -80°C. One week before implantation, cells were thawed and plated to reach 80-90% confluence on the day of implantation.Device

[0239] The device was composed of two parts: the cell chamber and a frame to protect cell chamber made with ABS material certified USP Class VI or ISO 10993-1 i.e. biomedical device. The device cell-chamber membrane confinement barrier was composed of a 0.4 pm semi-porous hydrophilic polypropylene membrane (Pall) covered by a 100 pm rigidifying polyester (RCT Reichel Chemitechnik) mesh. The device contained a lateral filling port for cell loading (a loading capacity of 100 pl) that was obscured after the loading and before implantation. The size of device is 15.45 x 26.91 x 0.77 mm (L x 1 x H). The implant was sterilized with ethylene oxide (EO) and let in desorption chamber for a week to remove EtO residues. The device was coated with GW2580 (HY-10917; MedChemExpress), an antifibrotic agent to limit the formation of fibrosis.Loading cells in the device

[0240] Cells were harvested with 0.05 % trypsin-EDTA solution (A3840401; Thermofisher) and resuspended in DMEM (Gibco) culture medium. Cells were resuspended in 50 pL DMEM culture medium. The cell suspension was mixed to 50 pL Geltrex (A1413201; Thermofisher,) on ice. An insulin syringe (324826; BD) was used to load devices. The device loading port was sealed with silicon (732; Dow Coming). Two concentrations of cells were loaded in device: either 250 000 cells or 1 million cells for hARSA or ApoE-hARSA. Devices with C2C12 non-transduced cells were loaded with 250 000 cells to be used as control mice non treated.Surgery

[0241] Thirty minutes before induction of anesthesia, analgesia (m el oxicam, 5 mg / kg, Boehringer Ingelheim) was administrated and was maintained three days after surgery. In addition, 10 pL / g of body weight of hydrant saline solution was injected subcutaneously, sterile eye ointment was applied. The surgical procedure was performed under isoflurane (IsoVet, lOOOmg / kg, Vetflurane, Virbac). Induction of anesthesia was 4 % isoflurane. Anesthetized mice were kept on a 37 °C heating blanket. The skin on the surgical site was shaved and disinfected with chlorhexidine 0.05 % (5 % Hibitane). An incision of 1 cm was performed in dorsal skin and subcutaneous skin of the back was cleared from muscle creating a surgical window. The subcutaneous tissue was spread with blunt-ended forceps to create a pocket for the implant. The device was placed subcutaneously. Subcutaneous tissue was sutured using resorbable suture (PGLA07CN; Vicryl 6.0; Vetsuture) and the incision with non-resorbable suture polypropylene (LENE1CN; Vetsuture). Animals were maintained at 37 °C during the postoperative recovery. Daily monitoring of weight and wound healing was performed.Design of experiment

[0242] We implanted in 6-month-old mice (symptomatic with sulfatide accumulation) and sacrificed them 3 months after implantation according to the groups as described below:(1) Wild-type mice i.e. Tg-ARSA+ / - mice: two groups sham mice (n=6) who have a surgery without device and mice implanted with C2C12 cells (i.e. non-transduced: NT; n=6)(2) Untreated mice i.e. Tg-ARSA- / - or MLD implanted with C2C12 cells (called MLD + device NT (non-transduced); n=5) to study the efficacy of treatment(3) Treated mice i.e. MLD (Tg-ARSA- / -) implanted with 250 000 (n=5) or 1 million (n=3-5) C2C12-LV-hARSA-HA (called MLD + device hARSA) or with 250 000 (n=5-8) or 1 million (n=5) C2C12-LV-ApoE-hARSA-HA (called MLD + device ApoE- hARSA).Tissue preparation

[0243] Three months after implantation, animals were euthanized by an intraperitoneal administration of a lethal dose of Euthasol (180 mg / kg, Vetcare). Mice were perfused intracardially with phosphate buffered saline (PBS). Brain, spinal cord, sciatic nerve, gallbladder, kidney, liver, spleen and skin were collected for analysis. Different structures of a cerebral hemisphere (cortex, cerebellum, pons and rest of the brain) were dissected and frozen in liquid nitrogen. Sciatic nerve and kidney were directly frozen in liquid nitrogen and stored in -80°C. For sulfatide analysis and protein extraction from the same samples, tissue samples were crushed in liquid nitrogen and divided into two equals parts. A cerebral hemisphere, a portion of spinal cord, sciatic nerve, kidney and gallbladder were post-fixed overnight in 4% paraformaldehyde (PFA) / PBS1X. Samples were rinsed in PBS IX and cryoprotected in 30% sucrose / PBSIX. Tissues were embedded in Tissue- Tek OCT compound (VWR International) and cut into 14-pm sagittal section (brain), transversal section (spinal cord), 4pm longitudinal section (sciatic nerve and kidney) or transversal section (gallbladder) using a cryostat (Leica, Langham, TX). Cryosections were dried at room temperature and stored at -80°C. A liver, spleen and skin were postfixed in PF A 4% prior to paraffin inclusion for histology (Cut 6-10 pm with microtome).Explantation device

[0244] Cells were collected from the explanted devices with an insulin syringe and placed in a 1.5-ml reaction tube. The latter is centrifuged at 1500 rpm for 5 min at room temperature. Each phase (cellular pellet and supernatant) was collected and is frozen at 80°C.Quantitative PCR

[0245] DNA was extracted from cells (NT, hARSA and ApoE-hARSA) using chloroform / phenol protocol. hARSA-vector genome copy numbers were measured by quantitative PCR in pellet cells using the Light Cycler 480 SYBR Green I Master (Roche, France) as described (Sevin C et al. Hum. Mol. Genet. 2006;15(l):53-64). The results (vector genome copy number per cell) were expressed as n-fold differences in the transgene sequence copy number relative to the Adck3 gene copy as internal standard (number of viral genome copy for 2N genome).ARSA expression and activity

[0246] Samples (tissue or cellular pellet (i.e. lysate) or supernatant from cell culture or liquid within device) were homogenized in 0.3 ml of lysis buffer (100 mM Trizma base, 150 mM NaCl, 0.3% Triton; pH 7), incubated for 30 min on ice and centrifuged. The supernatant was collected for the determination of (1) protein content (bicinchoninic acid [BCA] protein assay kit; Pierce Biotechnology / Thermo Fisher Scientific, Rockville, IL); and (2) ARSA activity, using the artificial p-nitrocatechol sulfate (pNCS) substrate assay (Sigma-Aldrich, France) (Piguet F et al., "Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate. Human Molecular Genetics", 2010 Jan 1, 19(1): 147-58). Assays were performed in triplicate and results are expressed as nanomoles of 4-nitrocatechol (4NC) per hour per milligram of protein. And (3) the concentration of recombinant hARSA using an indirect sandwich ELISA specific for human ARSA as described (Matzner et al. Gene Ther. 7, 1250-1257), using 2 specific antibodies. Assays were performed in duplicate, and results are expressed as nanograms of hARSA per milligram of protein. All samples were quantified in duplicates.Biocompatibility Assessment1. Post-surgery and necropsy score

[0247] A score assessing the device biocompatibility was applied for three days in postsurgery, at 7 days post-surgery and the day of necropsy integrating tissue inflammation, reopening of the wound, scratching. Data are reported on a scale with a maximum score of 24 points (worst condition) for the three first days of observation; a scale with a maximum of 8 points (worst condition) for 7 days of observation. The maximum score was 8 points (worst condition) for the macroscopy assessment of animals during the sacrifice. The criteria of splenomegaly was not used because this mouse model already have splenomegaly linked to their disease.2. Histological staining

[0248] Liver, spleen and skin sections were stained with Mayer’s hematoxylin (HE, C0303, DiaPath) and eosin using standard procedures to visualize the tissue morphology. Skin sections were stained with Masson’s tri chrome, using standard procedures tovisualize the tissue morphology. Mast cells are visualized by classical toluidine staining (i.e., toluidine 0.1% for 3 min). Cellular infiltration was assessed with DAPI. Slices were acquired at 20X by using a slide scanner (NanoZoomer2.ORS, Hamamatsu) or at 20X by using a slide scanner (Axioscan, Zeiss).3. Histological analysis

[0249] Different parameters of the tissue at the implantation site were quantified. The thickness of the epidermis and the muscle were measured in 6 areas of the skin section for each animal. The area of adipose tissue was measured in a minimum of 50 areas for each animal. The muscle degeneracy was assessed in 6 areas of the skin section for each animal. The number of nuclei bearing muscle fibers was enumerated for each area. Then a percentage of muscle fibers in neurodegeneration was calculated. The neovascularization of the devices was evaluated by Masson’s tri chrome staining. The number of blood vessels were counted at the implantation in 6 areas of the skin section for each animal. The number was related to the area of the region in mm2. A mean of each parameter by area was performed for each animal. For mast cells (toluidine staining) and cell infiltration (DAPI), the number of cells at the implantation was counted in 3 distinct areas, taken randomly on the skin section. The number of cells counted was reported to the area of counting in mm2. The average was made over the 3 values obtained for each mouse.Histopathology

[0250] To evaluate sulfatide storage, frozen sections were postfixed in 4% PF A, stained with Alcian blue (A5268; Sigma-Aldrich) (0.05% in 0.025 M sodium acetate buffer, pH 5.7, containing 0.3 M MgC12 and 1% PF A), rinsed in the same buffer without dye, counterstained with fast red (229113; Sigma- Aldrich) and mounted as previously described (Piguet F et al. Hum. Gene Ther. 2012;23(8):903-914).

[0251] Immunohistochemical labelling to study inflammation was performed with the ABC method, following the protocol already described (Audouard E et al. Front. Mol. Neurosci. 2021; 14:95). In some cases, slides were counterstained with hematoxylin. The slides were mounted with Eukitt (VWR International).

[0252] For immunofluorescence labelling, cells were fixed with PFA 4% and PBS IX for 15 min. After washes in PBS, cells were permeabilized with PBS / 0.3% TritonX-100 for 15 min and saturated with PBS / 0.1% Triton / 5% NGS for 30 min. Primary antibodies were diluted in saturation. Primary antibodies were diluted in the saturation solution and incubated Ih at 37°C. After wash in PBS / triton 0.1%, secondary antibodies were diluted in saturation buffer and incubated for Ih at 37°C. After washing with PBS, slides were mounted with DAPI Fluoromount (#0100-20; SouthernBiotech).

[0253] Primary antibodies used were the followings: mouse anti-GFAP (G3893, Sigma- Aldrich; 1 :400), rabbit anti-Ibal (019-19741, WAKO; 1 :750), rabbit anti-ARSA (1 : 1,000) and rat anti -Lamp 1 (1D4B, DSHB, 1 :200).

[0254] Secondary antibodies were: biotinylated goat anti-rabbit (BA- 1000, Vector Lab; 1 :1000), biotinylated goat anti-mouse (BA9200, vector Lab; 1 : 1000); goat anti-rabbit AlexaFluor 488 (Al 1034, Life technologies, 1 :1000); goat anti-rat AlexaFluor 594 (Al 1007, life technologies, 1 : 1000).

[0255] Images are acquired at 20X by using a slide scanner (Axioscan, Zeiss).Stereological cell counts

[0256] Stereological counts were performed by two-three independent investigators, using Image J or photoshop or Zen softwares. All quantifications were done on three sections for each tissue and for each animal. For Alcian staining, the number of sulfatide storage inclusions were quantified and reported to quantified area in mm2in three random areas of cortex, corpus callosum, fimbria, cerebellar white matter and sciatic nerve. For kidney, the number of sulfatide storage inclusions were quantified and reported to total number of structures and to quantified area in mm2in three random areas of cortex. A percentage of positive cells was calculated for each animal. For gallbladder, area of alcian blue staining on total epithelial cell area has been performed. A percentage of positive area was calculated for each animal. In the spinal cord, for GFAP, three random areas of spinal cord were counted. In the spinal cord, for sulfatide storage, a hemi-section was counted.

[0257] Stereological counts were performed by two-three independent investigators, using AIFORIA. For GFAP and Ibal labelling, the number of positive cells were evaluated in whole area of the brain section and were reported to quantified area in mm2. All results were assessed per mm2and expressed as the mean ±SEM.Statistical analysis

[0258] Data were analyzed using GraphPad Prism 8 software. The statistical significance of values among groups was evaluated by ANOVA, followed by the least significant difference t-test. All values used in figures and text are expressed as mean ±standard error of the mean (SEM). Differences were considered significant at p < 0.05.ResultsConstruction and validation of genetically engineered cells

[0259] To establish a stable cell line producing human ARSA, a lentiviral vector (LV) has been transduced into C2C12 cell lines. Two constructions were performed in parallel. The first lentiviral vector to target peripheral nervous system (PNS) and peripheral organs was LV-hARSA-HA. This LV was transduced at 50 MOI (multiplicity of infection) in C2C12 cell line. For the second construction, we integrated an ApoE-II derived signal peptide in frame with the hARSA-HA gene (ApoE-hARSA-HA). After validation of the construction by sequencing, a production of LV has been performed. This LV was transduced at 50 MOI in C2C12 cell line.

[0260] For each genetically engineered cell line, number of vector genome copy (VGC), ARSA activity and its expression were measured after transduction to validate their functionalities (Figures 1A-C). The mean number of VGC in C2C12-LV-hARSA-HA and C2C12-LV- ApoE-hARSA-HA (called hARSA and ApoE-hARSA to simplify the text) was of 2 and 0.5 VGC, respectively (Figure 1A), indicating the presence of recombinant hARSA in DNA of these cells (and meaning chromosomal integration of the recombinant hARSA protein in these cells). Moreover, 3-4 days after transduction of C2C12 cells, the culture medium supernatant and cell lysate have been collected to assess ARSA activity. ARSA activity in cell lysate or in culture medium supernatant of thesecell lines was superior to activity present in non-transduced C2C12 (Figure IB). Finally, ARSA expression was evaluated by ELISA using an antibody directed against hARSA in culture medium supernatant of these cell lines (collected some days after transduction of cells). The mean of its expression was 169 and 403 ng ARSA / mg protein in hARSA and ApoE-hARSA constructions respectively, whereas it was not detected in non-transduced C2C12 (Figure 1C). We demonstrated a high hARSA expression in these two cell constructions. To conclude, C2C12 transduced with hARSA or ApoE-hARSA express high levels of functional ARSA enzyme.

[0261] In the literature, ARSA enzyme is localized in lysosomes. To verify whether hARSA was perfectly transduced in C2C12 cells as well as localized in lysosome, an immunofluorescence was performed in co-staining with anti -hARSA and anti -Lamp 1 (lysosomal marker) antibodies on the different cell constructions. A colocalization of hARSA and Lampl was observed in cells transduced with hARSA or ApoE-hARSA, indicating that hARSA is correctly localized in lysosomes and thus could catabolize sulfatides. All these data validate these two genetically engineered cells, which could be tested like treatment against MLD.

[0262] To assess the capacity of cells to secrete ARSA in the time, devices loaded with cells containing either hARSA or ApoE-hARSA were put in culture during several weeks. Before each medium culture change, supernatant of each well containing a device was collected to assess its ARSA activity. ARSA activity was detected for each construction, indicating that recombinant hARSA protein was secreted in supernatant. After 80 days, cells and liquid within device were collected to measure ARSA activity to assess the functionality of cells. ARSA activity was observed in cells and liquid within device of both constructions, indicating that cells remain functional after several weeks of secretion. Thus, cells retain the capacity of hARSA secretion during several months.Characterization of loaded cells in devices

[0263] Additionally, to the validation of genetically engineered cells, it was important to ensure that cells implanted in animals were well functional, i.e. able to secrete hARSA. Therefore, the day where cells were loaded in device, the culture medium supernatant offlask containing cells and a pellet of cells were picked up at each experiment to assess ARSA activity and its expression. ARSA activity and its expression were well detected in the pellets of hARSA or ApoE-hARSA cells as well as in the supernatant of these cells in comparison to non-transduced cells (Figures 2A-B). Moreover, during the explantation, cells loaded in cell chamber as well as liquid within the device were collected to assess ARSA activity. This allow us to verify whether transduced cells were still functional and alive three months after implantation. For each group implanted with transduced cells, we detected ARSA activity well superior to non-transduced cells both in the cells and liquid within device (Figure 2C). All these data show that we have implanted animals with functional transduced cells (i.e. with the capacity to secrete hARSA) and that, three months after implantation, cells were still functional.The device was tolerated by implanted MLD mice

[0264] The encapsulated cell technology (ECT) was implanted for three months in MLD mice; it is primordial to assess whether the ECT is biocompatible and well-tolerated by implanted animals. The formation of fibrosis has been limited by the coating of device with antifibrotic agent, the GW2580. A score of biocompatibility was applied during the post-surgical period (Days 1 to 3 and 7 days) and another at necropsy. This score is an end-point to euthanize animals before the end of experiment, i.e. a score superior to 6 after 7 days of implantation is the sign of a problem in mouse. No animal was euthanized before the end of the experiment. The score of biocompatibility for the three first days after implantation was of 8 and 9 points (on maximum score of 24 points) in sham and implanted mice, respectively. The score was therefore similar between two groups. It is normal to observe inflammation after a surgery, hence this slightly high score. Then seven days after surgery, the score was of 1 and 3 points in sham and implanted mice, respectively. It is a good sign to see a decrease in the score 7 days after implantation, indicating a good healing of the wound. Moreover, during the necropsy, the score was of 0 or 1 point in sham and implanted mice, indicating a complete healing (Figure 3). We observed the same profile of score evolution when we distinguished mice implanted with non-transduced cells from those implanted with cells transduced with either hARSA or ApoE-hARSA. The score of 1 point during the necropsy was due to the formation of thinlayer of fibrotic tissue around the device.

[0265] Then, histological analysis of skin will allow us to assess the biocompatibility of device. The skin in contact with the device was collected during the necropsy for each group and we have performed several staining (Hematoxyline-eosin (HE) and Masson’s tri chrome) to assess it. Diverse parameters (i.e. thickness of fibrotic tissue, adipose tissue, muscular fiber, neovascularization) were measured on skin side to evaluate local immunological effects of implantations. Indeed, a thin layer of fibrotic tissue, an absence of adipose tissue dilation and muscle neurodegeneration as well as the presence of blood vessels near the device will be indicative of a reduced immunological response, sign of a biocompatible device in animal. The thickness of normal epidermis was significantly thinner in implanted MLD mice in comparison to sham groups (Figure 4A). The thickness of fibrotic tissue was identical between each implanted groups, except for implanted MLD mice with device containing hARSA cells, its thickness was significantly thinner than implanted MLD mice with C2C12 cells (Figure 4A). However, this observation was no longer present when we distinguished the number of cells loaded in the device between the two constructs of hARSA or ApoE-hARSA cells. The thickness of fibrotic tissue is relatively thin compared to that of the epidermis. Area of adipose tissue was similar between each group of implanted mice, except in implanted mice with device containing hARSA cells where area was significantly decreased (Figure 4B). Nevertheless, this observation was no longer present when we distinguished the number of cells loaded in the device between the two constructs of hARSA or ApoE-hARSA cells. Thus, the area of adipose tissue is not significantly increased compared to sham group (Figure 4B), indicating an absence of tissue damage. The percentage of muscle fibers in neurodegeneration was weak between each group of implanted mice (Figure 4C), indicating an absence of muscular degeneration. The number of blood vessels was significantly increased in implanted MLD mice, a sign of neovascularization of device (Figure 4D). All these data show a reduced immunological response.

[0266] Moreover, toluidine coloration was performed on the skin to quantify the number of mast cells and DAPI to score cellular infiltration. The number of mastocytes or cellular infiltration were similar between each group of implanted mice (Figures 4E-F). All thesedata show that the implantation of device does not create tissue damage nor severe inflammation and immune response. On the contrary, a neovascularization is formed around the device, a sign of well-tolerance.

[0267] Finally, hematoxylin-eosin staining was performed on liver and spleen section of each group to evaluate a potential toxicity of treatment. Histological analysis of liver and spleen section have shown similar pictures in each group.

[0268] To conclude, the device is biocompatible in MLD mice. h-ARSA secreting encapsulation cell technology ofhARSA secretion did not alter hARSA activity in the CNS or kidney of treated MLD mice

[0269] To assess whether the treatment (i.e. recombinant hARSA) is detectable in tissues of treated MLD mice, ARSA activity was measured in several structures of the CNS and in kidney (Figure 2D). ARSA activity was similar between WT and untreated mice, as expected. ARSA activity was significantly increased in cerebellum of treated MLD mice with 1 million ApoE-hARSA transduced cells in comparison to WT mice. On the other hand, ARSA activity was significantly decreased in pons of treated MLD mice with 1 million ApoE-hARSA transduced cells and in the kidney of treated MLD mice with 250 000 cells transduced with ApoE-hARSA in comparison to WT and / or untreated MLD mice. Otherwise, ARSA activity was similar in the other structures and groups of treated MLD mice. No excess hARSA was found in the tissues of treated mice. It is probably normal because the ECT, which allows the continuous secretion of hARSA, was directly used by the tissues to be treated.Significant improvement of MLD pathophysiology in implanted MLD mice with continuous-hARSA secreting ECT

[0270] We have decided to implant device in 6-month-old MLD mice because at this stage, mice already show characteristic symptoms of the disease, i.e. sulfatide storage and inflammation of CNS. To assess the therapeutic efficiency, two criteria that are the hallmarks of the disease were analyzed, namely sulfatide storage and neuroinflammation. a. the ECT led to reduce the sulfatide accumulations in CNS and sciatic nerve ofimplanted mice.

[0271] To assess whether the continuous-hARSA secreting ECT could decrease sulfatide storage, we used two different methods: Alcian Blue staining and GCMS analysis. Alcian Blue coloration was performed on the brain, spinal cord, sciatic nerve, gallbladder and kidney sections of untreated and treated MLD mice and compared to WT tissues. Nine-month-old untreated MLD mice display massive sulfatide storage in the brain, spinal cord, sciatic nerve, gallbladder and kidney, compared to WT mice (Figures 5 and 6). In treated mice with hARSA transduced cells, 3 months after implantation, hARSA secreting ECT led to a significant decrease of the number of sulfatide storage in the substructures of brain (Figures 5A-D). No significant difference of treatment has been observed between the two concentrations of hARSA cells in the brain. Moreover, a significant decrease in the number of sulfatide inclusions was observed in the brain of implanted mice with ApoE-hARSA transduced cells compared to untreated mice. This decrease was significantly more important in the corpus callosum, the fimbria and the cerebellum of implanted mice with 1 million than 250 000 ApoE-hARSA cells. In the spinal cord of treated mice, only implanted mice with 1 million hARSA cells showed a significant decrease of sulfatide storage compared to untreated MLD mice. In conclusion, the hARSA-secreting ECT and ApoE-hARSA-secreting ECT are both efficient therapies in the CNS of treated mice. Moreover, the number of sulfatide accumulations was significantly decreased in treated mice with 1 million hARSA cells and 250 000 ApoE- hARSA cells in sciatic nerve; and we observe a non-significant decrease in other groups of treated mice (Figure 6). For gallbladder and kidney, no change in the percentage of positive cells / areas has been shown in different groups of treated MLD mice in comparison to untreated MLD mice. To conclude, the hARSA-secreting ECT and ApoE- hARSA-secreting ECT reduce sulfatide accumulations in PNS of treated mice. b. the ECT led to reduce neuroinflammation in CNS of implanted mice

[0272] To assess the effect of continuous-hARSA secreting ECT on the MLD mice neuroinflammation, an immunohistochemical staining was performed to evaluate astrogliosis and microgliosis on the brain and spinal cord sections of the different groups of mice. A significant increase of Ibal- and GFAP-positive cells was observed in thebrain and the spinal cord of untreated MLD mice at 9 months, compared to WT mice. After implantation of hARSA or ApoE-hARSA secreting ECT, a significant correction of microgliosis was observed in the whole brain of treated mice (Figure 7). In the spinal cord, only implanted mice with 1 million ApoE-hARSA cells showed a significant decrease of microglia number in comparison untreated MLD mice (data not shown). No correction of astrogliosis has been observed in the brain of treated mice in comparison to untreated mice. Only implanted mice with hARSA showed a significant correction of astrogliosis in the spinal cord in comparison to untreated mice (data not shown).

[0273] To conclude, a continuous-hARSA or ApoE-hARSA-secreting ECT in MLD mice showed a significant correction of sulfatide accumulations in nervous system (CNS and sciatic nerve) as well as of the neuroinflammation in CNS in treated MLD mice.Example 2: Cell-based therapy in non-human primates1. First experimental design (subcutaneous implantation)Materials and MethodsAnimal Care

[0274] Non-human primates (Macaca fascicularis) were maintained in a temperature- and humidity-controlled animal facility (target temperature 20-24QC, with a 12-h light — dark cycle. NHP diet pellets were given ad libitum except during the fasting experimental period. At least 1 fruit and 1 vegetable per day and supplement of seeds or dry grapes were given to animals. In addition, according to animal welfare, each animal had access to enrichment in the cage, balls, scale, etc and once a week a special enrichment (swimming pool, video, searching like straw with popcorn inside) was offered. Tap water was offered ad libitum in polycarbonate bottles. An acclimatization period of two weeks was observed when animals arrive in the animal facility prior to any manipulation.Implant design

[0275] The implantable capsule comprises a cell receiving portion comprising a porous membrane surrounding a cell receiving chamber for receiving immortalized cells in aliquid media, and a cell support matrix comprising at least one yam inserted within the cell receiving chamber configured for the arrangement of the immortalized cells within the cell receiving chamber. See an example of the implantable capsule in Figure 11. Each capsule was loaded with 30 mL of the cell suspension containing 1 M of ARSA fused to a HA-tag (ARSA-HA) secreting cells through a loading tube. After loading, the catheter was cut and capsules were sealed with UV-curable adhesive. Capsules were placed in 12- well plates containing 2 mL of culture medium per well and maintained in a humidified incubator with 5% CO2 at 37°C.

[0276] The secreting cells are immortalized human myoblast cells, and in particular, cells from the immortalized human myoblast cell line deposited with CCOS under accession number 1902.Experimental Design

[0277] One female NHP received 5 capsules containing each one 1 million ARSA-HA secreting cells.

[0278] Implants were placed in medium without FBS and rinsed in PBS for few minutes just prior to implantation. An intravenous catheter was placed in the caudal vein to maintain animal under Glucose and Ringer solution for hydration all along the procedure. Animal was placed under monitoring and temperature were maintained on a special blanket. After shaving, disinfection was done according to our standard protocol in the upper back of the animal close to the shoulder. Surgeon weas dressed in sterile coat and gloves and implantation of the 5 capsules was done sequentially using sterile material and proper device. Once implantation was done, sutures were done if necessary and animal was disinfect using aqueous chlorhexidine and vetramil was applied to allow proper cicatrization. Animal was placed back in the cage and feed once recover from the anesthesia. Animal was maintained isolated for a night and back in couple at day 1 upon verification of the wound.

[0279] Cerebrospinal fluid (CSF) samples were collected at Baseline and then day 4 (D4), 3 weeks and 6 weeks frozen in 2 aliquots and additional pellet for CSF were frozen.

[0280] ARSA activity was measured in CSF. Samples were collected for the determination of (1) protein content (bicinchoninic acid [BCA] protein assay kit; Pierce Biotechnology / Thermo Fisher Scientific, Rockville, IL); and (2) ARSA activity, using the artificial p-nitrocatechol sulfate (pNCS) substrate assay (Sigma-Aldrich, France) (Bass NH et al. 1970 Jan;20(l):52-62.). Assays were performed in triplicate and results are expressed as nanomoles of 4-nitrocatechol (4NC) per hour per milligram of protein.Results

[0281] ARSA activity was measured in CSF from one non-human primate having received 5 capsules containing each one 1 million ARSA-HA secreting cells.

[0282] Figure 8 shows that an increased ARSA activity was detected in the primate as soon as 6 weeks post implantation, demonstrating that the implant is able to release ARSA in the CSF of non-human primates.2. Second experimental design (subdural implantation)Materials and Methods

[0283] A scale up pilot study on two non-human primates (NHP) was realized to define the parameters for having sufficient ARSA infusion in patients to treat the disease using subdural implantation.

[0284] For this second experimental design, two NHP (Macaca fascicularis) were maintained in the same animal care conditions as previously mentioned, with an acclimatization period of two weeks.

[0285] The implantable capsule used for the second experimental design was the same as in the first experimental design, except that the capsule was shortened to avoid damaging parenchyma tissues (the extractor portion of the capsule in Figure 11 was removed).. In this second experimental design, two types of implantable capsules were used: implantable capsules containing ARSA-HA secreting cells or implantable capsules containing ApoE- ARSA-HA secreting cells. The secreting cells were the same as previously mentioned (cells from the immortalized human myoblast cell line depositedwith CCOS under accession number 1902).

[0286] The first and second NHP received, by subdural implantation, 3 capsules containing each one 1 million of ARSA-HA secreting cells and 3 capsules containing each one 1 million of ApoE-ARSA-HA secreting cells, respectively.

[0287] Implants were placed in medium without FBS and rinsed in PBS for few minutes just prior to implantation. An intravenous catheter was placed in the caudal vein to maintain animals under Glucose and Ringer solution for hydration all along the procedure. Animals were placed under monitoring and temperature was maintained thanks to a special blanket. After shaving, disinfection was done on all the surface of the skull of the animals. Medtronic StealthStation S8 was used to have real time navigation based on the pre op MRI and to decide the best sites of implantation. Surgeons were dressed in sterile coat and gloves, and implantation of the 3 capsules was done sequentially using sterile material and proper device. Once implantation was done, sutures were done if necessary and animals were disinfected using aqueous chlorhexidine and vetramil was applied to allow proper healing. Animals were placed back in the cage, and feed once recover from the anesthesia. Animals were maintained isolated for a night and back in couple at day 1 upon verification of the wound.

[0288] Figure 14 shows the study design of this secondary experimental design, wherein the dark tubes are for blood sampling and the white tubes for cerebrospinal fluid sampling.

[0289] The 3 capsules containing ARSA-HA secreting cells or ApoE-ARSA-HA secreting cells were implanted at Day 0 (DO). Cerebrospinal fluid (CSF) samples and blood samples were collected at baseline, i.e. 7 days before DO (D-7), after 1 week and then were collected monthly, after 1 month (IM), after 2 months (2M), after 3 months (3M), after 4 months (4M) frozen in 2 aliquots and additional pellet for CSF were frozen. At the end, a necropsy was done at 5 months as show in Figure 14.

[0290] ARSA activity was evaluated in the collected samples with the same protocol as previously mentioned. Assays were performed in triplicate and results are expressed as nanomoles of 4-nitrocatechol (4NC) per hour per milligram of protein.

[0291] For both NHP, ARSA activity was measured: in cerebrospinal fluid (CSF) over in life (Figure 15) in central nervous system (CNS) in most brain regions, at necropsy (Figure 16A) in spinal cord and peripheral nervous system at necropsy (Figure 16B), and, in peripheral tissues at necropsy (Figure 16C).Results

[0292] No adverse effect of the treatment was observed in the two animals. They both add a 5-day period of recovery after the surgery, with less movement in the cage and decrease of appetite and food ingestion that was also manage with drugs. Normal behavior was observed in both animals mainly with feeding behavior, displacement in the cage and individual behavior.

[0293] Figure 15 shows that an increased ARSA activity was detected in the CSF of both primates as soon as 2 months post implantation, demonstrating that the subdural implant can release functional ARSA in the CSF of non-human primates.

[0294] Figure 16A shows that an increased ARSA activity was detected, in both primates, in CNS, in most brain regions, except substantia nigra, demonstrating that the implant can release functional ARSA in the CNS of non-human primates.

[0295] Figure 16B shows that an increased ARSA activity was detected, in both primates, in peripheral nerves and in spinal cord, demonstrating that the implant can release functional ARSA in the peripheral nerves and in spinal cord of non-human primates.

[0296] Figure 16C shows that ARSA activity was detected, in both primates, in peripheral tissues.3. Third experimental designMaterials and Methods

[0297] The third experimental design was done with two NHP, with subcutaneousimplantation of up to 10 capsules containing each one 1 million of ARSA-HA expressing cells or ApoE- ARSA-HA expressing cells in each NHP.

[0298] For this third experimental design, two NHP (Macaca fascicularis) were maintained in the same animal care conditions as previously mentioned, with an acclimatization period of two weeks. The capsules and expressing cells used were the same as in the first experimental design.

[0299] Figure 17 shows the design of subcutaneous implant study in NHP with the timeline of sample collection and implantation of the 10 capsules.

[0300] The 10 capsules were implanted subcutaneously with the same protocol as previously mentioned, with 5 capsules implanted at DO and 5 other capsules implanted 3 months after DO.

[0301] In this third experimental design, cerebrospinal fluid (CSF) samples were collected at baseline, i.e. 15 and 7 days before DO (D-15 and D-7), and then 1 week, 4 weeks, 6 weeks, 2 months, 3 months, 13 weeks, 4 months and 18 weeks after the first day of implantation (DO) frozen in 2 aliquots and additional pellet for CSF were frozen. At the end, a necropsy was done at 5 months.

[0302] ARSA activity was evaluated with the same protocol as previously mentioned. Assays were performed in triplicate and results are expressed as nanomoles of 4- nitrocatechol (4NC) per hour per milligram of protein.

[0303] For both NHP, ARSA activity was measured: in central nervous system (CNS) in most brain regions, at necropsy (Figure 18A) in spinal cord and peripheral nervous system at necropsy (Figure 18B).Results

[0304] The subcutaneous implantation has proven to be safe and well tolerated with up to 10 capsules, no change in body weight nor blood parameters variation were observed over the 5 months treatment.

[0305] Figure 18A shows that an increased ARSA activity was detected, in both primates, in the cortex, in pons, in the corpus callosum, and in the cerebellum.

[0306] Figure 18B shows that an increased ARSA activity was detected, in both primates, in the peripheral nerves and, to a lesser extent, in the spinal cord. An increase in ARSA activity was also detected in the DRG of one monkey.(Original in Electronic Form)(This sheet is not part of and does not count as a sheet of the international application)FOR RECEIVING OFFICE USE ONLYFOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. A genetically engineered cell secreting a protein of interest for use in treating a disease or condition affecting the nervous system in a subject in need thereof, wherein said cell is encapsulated in an implantable device and wherein said disease or condition affecting the nervous system is either: i) a storage disease, such as a lysosomal storage disease, and the protein of interest is the wild-type version of the mutated protein causing the storage disease, or ii) the disease or condition is Friedreich ataxia, and the protein of interest is frataxin.

2. The genetically engineered cell for use according to claim 1, wherein said disease or condition and said protein of interest are one of the followings combinations: i) the disease or condition is metachromatic leukodystrophy (MLD), and the protein of interest is arylsulfatase A (ARSA); ii) the disease or condition is a mucopolysaccharidosis and the protein of interest is an enzyme involved in the degradation of glycosaminoglycans, preferably the disease or condition is a mucopolysaccharidosis (MPS) type III, in particular either : a. MPS type III B, and the protein of interest is N-acetyl-alpha- glucosaminidase; or b. MPS type III A, and the protein of interest is N-sulfoglucosamine sulfohydrolase; iii) the disease or condition is Friedreich ataxia, and the protein of interest is frataxin.

3. The genetically engineered cell for use according to any one of claims 1 to 2, wherein the protein of interest is fused with one or several peptide(s) promoting protein secretion, protein recapture and / or protein addressing to cellular compartments.

4. The genetically engineered cell for use according to any one of claims 1 to 3, wherein the disease or condition is metachromatic leukodystrophy (MLD) and the protein of interest is ARSA, preferably human ARSA.

5. The genetically engineered cell for use according to claim 4, wherein ARSA is fused to one or several peptide(s) promoting protein secretion, protein recapture and / or protein addressing selected from the group comprising or consisting of a protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep-2 peptide (Ang-2), receptor-binding domains of human apolipoprotein B (ApoB) and ApoE, including ApoE-I and ApoE-II, a native secreted alkaline phosphatase (SEAP) secretion peptide, signal peptides derived from interleukins, and variants thereof, preferably wherein ARSA is fused to a receptor-binding domain of ApoE II.

6. The genetically engineered cell for use according to any one of claims 1 to 5, wherein the subject is symptomatic.

7. The genetically engineered cell for use according to any one of claims 1 to 6, wherein the subject has received or is to be received a transplantation of lentiviral corrected hematopoietic stem cells secreting the protein of interest in the brain.

8. The genetically engineered cell for use according to any one of claims 1 to 7, wherein the genetically engineered cell is an immortalized human myoblast cell or a progeny thereof derived from primary human myoblast cells.

9. The genetically engineered cell for use according to any one of claims 1 to 8, wherein the genetically engineered cell is a cell from the immortalized human myoblast cell line deposited with CCOS under accession number 1902.

10. The genetically engineered cell for use according to any one of claims 1 to 9, wherein the implantable device is an implantable capsule.

11. The genetically engineered cell for use according to claim 10, wherein the implantable capsule comprises a cell receiving portion comprising a porous membrane surrounding a cell receiving chamber for receiving immortalized cellsin a liquid media, and a cell support matrix comprising at least one yam inserted within the cell receiving chamber configured for the arrangement of the immortalized cells within the cell receiving chamber.

12. The genetically engineered cell for use according to any one of claims 1 to 11, wherein the implantable device comprises from about 100 000 to about 2 million of cells, preferably from about 150 000 to about 1.5 million of cells, more preferably from about 250 000 to about 1 million of cells.

13. The genetically engineered cell for use according to any one of claims 1 to 12, wherein the implantable device is adapted to be implanted subcutaneously, intramuscularly, intradermally, intravitreally, subdurally, intraparenchymally or within the intracerebroventricular fluid or within the cerebrospinal fluid in the subarachnoid space, preferably wherein the implantable device is adapted to be implanted subdurally.

14. A method for treating a disease or condition affecting the nervous system in a subject in need thereof, the method comprising:• providing an implantable device comprising genetically engineered cells secreting a protein of interest, wherein said cells are encapsulated in said implantable device; and• implanting said device subdurally in one or more locations selected from cerebral hemispheres, cisterna magna, and dorsal root ganglia from said subject;• wherein the disease or condition affecting the nervous system is either: i) a storage disease, such as a lysosomal storage disease, and the protein of interest is the wild-type version of the mutated protein causing the storage disease, or ii) Friedreich ataxia, and the protein of interest is frataxin; and• wherein the implantable device comprises from about 100,000 to about 2 million of said genetically engineered cells.