WO2025157848A1 - Methods for the intravenous treatment of sanfilippo syndrome type iiib - Google Patents

Abstract

The present invention relates to the treatment of Sanfilippo syndrome type B (MPS3B). In this study, the inventors developed a new approach by using AAVMacPNS1 vector encoding NAGLU and only intravenous (IV) injection. This new approach allows to a good transduction of the nervous system (central and peripheral nervous systems) and notably neurons, a good persistence, expression and release of the therapeutic protein in the systemic system and in the brain without immune reaction. This method is less invasive and less toxic since no injection is done in the brain parenchyma and thus can be used to treat baby and young children. Thus, the present invention relates to a vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N-acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject intravenously.

Description

METHODS FOR THE INTRAVENOUS TREATMENT OF SANFILIPPO

SYNDROME TYPE IIIB

FIELD OF THE INVENTION:

The present invention relates to a vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N- acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject intravenously.

BACKGROUND OF THE INVENTION:

Mucopolysaccharidosis type IIIB syndrome (also known as Sanfilippo syndrome type B) is a rare autosomal recessive lysosomal storage disorder with predominant neurological manifestation in affected children. It is caused by mutations in the a-N-acetylglucosaminidase (NAGLU) gene, coding for a lysosomal enzyme required for the stepwise degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of incompletely degraded GAGs in affected cells and extracellular spaces leads to cognitive retardation and further neurodegeneration of the central nervous system, leading to progressive deterioration of cognitive abilities before the age of 5 years, including language acquisition delay, cognitive delay and/or abnormal behaviour, and premature death in the second decade (1-4). The challenge to treat MPS IIIB syndrome lies in the design of a therapy to supply the missing enzyme to the brain as early as possible after birth. Several recent human gene therapy trials for the treatment of neurodegenerative diseases relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain (5-7). In preclinical studies in MPS IIIB mice (8, 9) and dogs (10, 11), beneficial biochemical and neurological effects with intracerebral gene therapy administered via a recombinant AAV vector encoding NAGLU were observed, associated with the release of therapeutic enzyme from transduced brain cells. These results led to the assessment of safety and efficacy of a novel intracerebral therapy in a phase 1/2 uncontrolled clinical trial, in which four children with MPSIIIB syndrome were enrolled to receive intraparenchymal deposits of a recombinant AAV vector serotype 2/5 (rAAV2/5) encoding human NAGLU combined with immunosuppression (ClinicalTrials.gov Identifier: NCT03300453). An intermediate report at 30months concluded that treatment was well tolerated and induced sustained enzyme production in the brain. Good tolerance, sustained NAGLU production and milder disease in the patient treated at very early stage were confirmed after a 5.5 year follow-up (Deiva et al. Hum Gene Ther. 2021 Oct;32(19-20): 1251-1259. doi: 10.1089/hum.2021.135).

However, the neurocognitive benefit was in all cases only partial, presumably because enzyme was not delivered to the periphery, leaving meninges and brain capillaries severely affected. Thus, there is still need of well tolerated, safe and efficient method to treat the patient suffering from Sanfilippo syndrome type B.

SUMMARY OF THE INVENTION:

In this study, the inventors developed a new approach by using AAVMacPNSl vector encoding NAGLU and only intravenous (IV) injection. They showed that this new approach allows to a good transduction of the nervous system (central and peripheral nervous systems) and notably neurons, a good persistence, expression and release of the therapeutic protein in the systemic system and in the brain without immune reaction. More, this method is less invasive and less toxic since no injection is done in the brain parenchyma and thus can be used to treat baby and young children.

Thus, the invention relates to a vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N- acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject intravenously.

Particularly, the invention is defined by its claims.

DETAILED DESCRIPTION OF THE INVENTION:

The NAGLU vector and used thereof

A first object of the invention relates to a vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N-acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject intravenously.

According to the invention, an administration of the vector in the venous system (intravenously) allows a targeting of the nervous system and enough enzyme activity in the affected cells. Surprisingly, the inventor showed that the administration of the vector (particularly the AAVMacPNSl) allows a very significant increase of NAGLU in different part of the brain without intracerebral injection.

As used herein, the term “Sanfilippo syndrome type B” also known as “mucopolysaccharidosis type IIIB” or “MPSIIIB” or “MPS3B” has its general meaning in the art and denotes a rare autosomal recessive lysosomal storage disease that primarily affects the brain and spinal cord. It is caused by the defect in the catabolism of large sugar molecules called glycosaminoglycans (GAGs, or mucopolysaccharides) in the lysosomes. The MPS3B is caused by mutations in the lysosomal NAGLU gene.

As used herein, the term “NAGLU” for “N-acetylglucosaminidase, alpha” has its general meaning in the art and denotes an enzyme that degrades heparan sulfate (a GAG subtype) by hydrolysis of terminal N-acetyl-D-glucosamine residues in N-acetyl-alpha-D- glucosaminide. Its Entrez reference number is 4669 (SEQ ID NO: 1) and its uniProt reference number P54802 (SEQ ID NO: 2). The two sequences described below relate to the human NAGLU.

SEQ ID NO: 1 : atggaggcggtggcggtggccgcggcggtgggggtccttctcctggccggggccgggggcgcggcaggcgacgagg cccgggaggcggcggccgtgcgggcgctcgtggcccggctgctggggccaggccccgcggccgacttctccgtgtcggtggagc gcgctctggctgccaagccgggcttggacacctacagcctgggcggcggcggcgcggcgcgcgtgcgggtgcgcggctccacgg gcgtggcggccgccgcggggctgcaccgctacctgcgcgacttctgtggctgccacgtggcctggtccggctctcagctgcgcctgc cgcggccactgccagccgtgccgggggagctgaccgaggccacgcccaacaggtaccgctattaccagaatgtgtgcacgcaaag ctactctttcgtgtggtgggactgggcccgctgggagcgagagatagactggatggcgctgaatggcatcaacctggcactggcctgg agcggccaggaggccatctggcagcgggtgtacctggccttgggcctgacccaggcagagatcaatgagttctttactggtcctgcct tcctggcctgggggcgaatgggcaacctgcacacctgggatggccccctgcccccctcctggcacatcaagcagctttacctgcagc accgggtcctggaccagatgcgctccttcggcatgaccccagtgctgcctgcattcgcggggcatgttcccgaggctgtcaccagggt gttccctcaggtcaatgtcacgaagatgggcagttggggccactttaactgttcctactcctgctccttccttctggctccggaagacccc atattccccatcatcgggagcctcttcctgcgagagctgatcaaagagtttggcacagaccacatctatggggccgacactttcaatgag atgcagccaccttcctcagagccctcctaccttgccgcagccaccactgccgtctatgaggccatgactgcagtggatactgaggctgt gtggctgctccaaggctggctcttccagcaccagccgcagttctgggggcccgcccagatcagggctgtgctgggagctgtgccccg tggccgcctcctggttctggacctgtttgctgagagccagcctgtgtatacccgcactgcctccttccagggccagcccttcatctggtgc atgctgcacaactttgggggaaaccatggtctttttggagccctagaggctgtgaacggaggcccagaagctgcccgcctcttccccaa ctccaccatggtaggcacgggcatggcccccgagggcatcagccagaacgaagtggtctattccctcatggctgagctgggctggcg aaaggacccagtgccagatttggcagcctgggtgaccagctttgccgcccggcggtatggggtctcccacccggacgcaggggcag cgtggaggctactgctccggagtgtgtacaactgctccggggaggcctgcaggggccacaatcgtagcccgctggtcaggcggccg tccctacagatgaataccagcatctggtacaaccgatctgatgtgtttgaggcctggcggctgctgctcacatctgctccctccctggcca ccagccccgccttccgctacgacctgctggacctcactcggcaggcagtgcaggagctggtcagcttgtactatgaggaggcaagaa gcgcctacctgagcaaggagctggcctccctgttgagggctggaggcgtcctggcctatgagctgctgccggcactggacgaggtg ctggctagtgacagccgcttcttgctgggcagctggctagagcaggcccgagcagcggcagtcagtgaggccgaggccgatttctac gagcagaacagccgctaccagctgaccttgtgggggccagaaggcaacatcctggactatgccaacaagcagctggcggggttggt ggccaactactacacccctcgctggcggcttttcctggaggcgctggttgacagtgtggcccagggcatccctttccaacagcaccagt ttgacaaaaatgtcttccaactggagcaggccttcgttctcagcaagcagaggtaccccagccagccgcgaggagacactgtggacct ggccaagaagatcttcctcaaatattacccccgctgggtggccggctcttggtga

SEQ ID NO: 2:

MEAVAVAAAVGVLLLAGAGGAAGDEAREAAAVRALVARLLGPGPAADFSV SVERALAAKPGLDTYSLGGGGAARVRVRGSTGVAAAAGLHRYLRDFCGCHVAWSG SQLRLPRPLPAVPGELTEATPNRYRYYQNVCTQSYSFVWWDWARWEREIDWMALN GINLALAWSGQEAIWQRVYLALGLTQAEINEFFTGPAFLAWGRMGNLHTWDGPLPP SWHIKQLYLQHRVLDQMRSFGMTPVLPAFAGHVPEAVTRVFPQVNVTKMGSWGHF NCSYSCSFLLAPEDPIFPIIGSLFLRELIKEFGTDHIYGADTFNEMQPPSSEPSYLAAATT AVYEAMTAVDTEAVWLLQGWLFQHQPQFWGPAQIRAVLGAVPRGRLLVLDLFAES QPVYTRTASFQGQPFIWCMLHNFGGNHGLFGALEAVNGGPEAARLFPNSTMVGTGM APEGISQNEVVYSLMAELGWRKDPVPDLAAWVTSFAARRYGVSHPDAGAAWRLLL RSVYNCSGEACRGHNRSPLVRRPSLQMNTSIWYNRSDVFEAWRLLLTSAPSLATSPA FRYDLLDLTRQAVQELVSLYYEEARSAYLSKELASLLRAGGVLAYELLPALDEVLAS DSRFLLGSWLEQARAAAVSEAEADFYEQNSRYQLTLWGPEGNILDYANKQLAGLVA NYYTPRWRLFLEALVDSVAQGIPFQQHQFDKNVFQLEQAFVLSKQRYPSQPRGDTVD LAKKIFLKYYPRWVAGSW

As used herein, the term “periphery” denotes that the vector of the invention is administrated into the circulatory system and but also in the nervous system as the vector crosses the blood brain barrier (BBB) and notably in cerebellum, spinal cord, peripheral nerves and DRG and also in the brain grey nuclei, white matter including internal capsules and corpus callosum) but more diffusely and with a very homogenous biodistribution. According to the invention, the vector of the invention is particularly administrated intravenously. Thus, the invention relates to a vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N- acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid wherein the vector is administrated intravenously.

As used herein, the term “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed or translated.

As used herein, the terms “coding sequence” or “a sequence which encodes a particular protein” or “encoding nucleic acid”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

In a preferred embodiment, the invention provides a nucleic acid construct comprising sequence SEQ ID N°1 or a variant thereof for use in the treatment of Sanfilippo syndrome type B (MPS3B).

The variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc. The term variant also includes NAGLU gene sequences from other sources or organisms. Variants are preferably substantially homologous to SEQ ID No 1, i.e., exhibit a nucleotide sequence identity of typically at least about 75%, particularly at least about 85%, more particularly at least about 90%, 91%, 02%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID No 1. Variants of a NAGLU gene also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions. Typical stringent hybridisation conditions include temperatures above 30° C, preferably above 35°C, more preferably in excess of 42°C, and/or salinity of less than about 500 mM, preferably less than 200 mM. Hybridization conditions may be adjusted by the skilled person by modifying the temperature, salinity and/or the concentration of other reagents such as SDS, SSC, etc. As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject according to the invention is a human. Particularly, a subject according to the invention is a human with a Sanfilippo syndrome type B (MPS3B).

As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness (e.g., the pattern of dosing used during therapy). A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The invention also relates to a method of treating Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a vector which comprises the full sequence of N- acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject in the venous system. As used herein, the term “vector” has its general meaning in the art and refers to the vehicle by which a nucleic acid molecule can be introduced into cells, to transform the cell and promote expression (e.g. transcription and/or translation) of the introduced sequence. According to the invention, vectors include viral vectors or non-viral vectors.

Non-viral vectors

In a preferred embodiment, the vector use according to the invention is a non-viral vector. Typically, the non-viral vector may be a plasmid encoding NAGLU.

Particularly, a non-viral vector can be an exosome.

Non-viral vectors mainly comprise chemical systems that are not of viral origin and generally include chemical methods such as cationic liposomes and polymers. Non-viral vectors useful in the practice of the present invention has very well known in the art. According to the invention, non-viral vectors include but are not limited to liposomes such as cationic liposomes, solid-lipid nanoparticles (SLNs or LNPs) such as [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM- 102-based nanonparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al. Biol Pharm Bull (2018). Non-viral vectors according to the invention include also the non-viral vectors described in patent WO2017049245 and W02018081480.

In some embodiments, the method according to the invention, wherein the non-viral vector is cationic a polymers-based nanoparticle, and more particularly is a poly ethyl enimine(PEI)-b ased nanoparti cl e .

In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) encoding NAGLU.

In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding NAGLU. Viral vectors

Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.

The terms “Gene transfer” or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e. g. , episomes), or integration of transferred genetic material into the genomic DNA of host cells.

Examples of viral vector include adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94/19478.

In a particular embodiment, adeno-associated viral (AAV) vectors are employed.

In another particular embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO and all variants of AAV9, including AAV PHP.B (see for example the patent application WO2015038958), AAVPHP.eB, AAV-PHP.N", and "AAV-PHP.B- DGT (see the patent application W02017100671 or Chan Y Ken, Nat Neurosci. 2017 Aug;20(8): 1172-1179.), AAV3B, AAV-2i8, Rh74, AAV capBlO, AAVMacPNSl or AAVMacPNS2 or any other serotypes of AAV that can infect human, nonhuman primates (NHP) or other species.

In a more particular embodiment, the AAV vector is an AAVMacPNSl or an AAVMacPNS2 or any vector upgrade from the MacPNSl and able to better cross the BBB.

Thus, the invention relates to a vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N- acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid wherein the vector is administrated to the said subject in the periphery and wherein the vector is the AAVMacPNSl or AAVMacPNS2.

As used herein the term “AAVMacPNSl” or “AAVMacPNS2” denotes an optimized AAv serotype that is able to cross the Blood brain barrier after intravenous delivery. These AAV were notably engineered for non-invasive gene delivery to rodent and non-human primate nervous systems.

Nucleic acids sequence of the AAVMacPNSl (SEQ ID NO:3):

GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCCCAATGATACG CGTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACG ACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACATCG ACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCG CAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGA CGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAA TGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCC TGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGA GTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTT CCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGT TTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGC CGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCG GGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCA GCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTG AATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAG ACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATC AGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAG GGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCT TCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGG AAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCC GTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACG ATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAA GAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGC GGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAG AACTTTCCCTTCAACGACTGTGTGGACAAGATGGTGATCTGGTGGGAGGAGGGGA

AGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGG

TGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGAT

CGTCACCTCCAACACCAATATGTGCGCCGTGATTGACGGGAACTCAACGACCTTC

GAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTC

TGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG

GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGG

AGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGT

GCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTAC

GCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGT

TTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCA

CGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTG

TCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGT

GCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTT

TCTGAACAATAAATGACTTAAACCAGGTATGAGTCGGCTGGATAAATCTAAAGTC

ATAAACGGCGCTCTGGAATTACTCAATGAAGTCGGTATCGAAGGCCTGACGACA

AGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTG

AAGAACAAGCGGGCCCTGCTCGATGCCCTGGCCATCGAGATGCTGGACAGGCAT

CATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACA

ACGCCAAGTCATTCCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCA

TCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGC

GTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCC

GTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCA

AAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAA

GCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGG

AACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGG

CCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGA

CTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGC

TCCCCGGGTAAATGCATGAATTCGATCTAGAGGGCCCTATTCTATAGTGTCACCT

AAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCT

GTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGT

CCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA

TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAAT AGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACC

AGCTGGGGCTCGAATCAAGCTATCAAGTGCCACCTGACGTCTCCCTATCAGTGAT

AGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAG

AACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTG

ATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGT

CTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGT

GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCC

TATATAAGCAGAGAGATCTGTTCAAATTTGAACTGACTAAGCGGCTCCCGCCAGA

TTTTGGCAAGATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAAAGGTC

AATCAGGTGCCGGTGACTCACGAGTTTAAAGTTCCCAGGGAATTGGCGGGAACTA

AAGGGGCGGAGAAATCTCTAAAACGCCCACTGGGTGACGTCACCAATACTAGCT

ATAAAAGTCTGGAGAAGCGGGCCAGGCTCTCATTTGTTCCCGAGACGCCTCGCAG

TTCAGACGTGACTGTTGATCCCGCTCCTCTGCGACCGCTAGCTTCGATCAACTACG

CAGACAGGTACCAAAACAAGTGTTCTCGTCACGTGGGCATTAATCTGATTCTGTT

TCCCTGCAGACAATGCGAGAGAATGAATCAGAACTCAAATATCTGCTTCACTCAC

GGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTG

TCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGT

GCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATC

TTTGAACAATAAATGACTTAAGCCAGGTATGGCTGCCGATGGTTATCTTCCAGAT

TGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTG

GAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTAGAGGTCTTG

TGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGC

CGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAAGCCTACGACCAGC

AGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGT

TCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAG

TCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGC

TAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGA

CTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAAT

TTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAA

CCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCG

CACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAA

ATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCG

AACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGC ACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGG

GGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCG

ACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTT

AACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAAT

AACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACG

TGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCAT

GATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGT

TCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACA

ACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCA

CAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTAT

CTCTCTAGAACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTG

TGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCA

GCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATT

TGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATC

CTGGACCTGCTATGGCCTCTCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCT

GGATCTTTAATTTTTGGCAAACAAGGTACTGGCAGAGACAACGTGGATGCGGACA

AAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGG

AGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAACCCCACGAAGGCA

GCAGCAGAGCACAGGCGCAGACCGGTTGGGTTCAAAACCAAGGAATACTTCCGG

GTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAAT

TCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATG

AAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTC

CAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGG

TCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTG

GAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTT

GCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACC

TGACTCGTAATCTGTAAGTCGACTTGCTTGTTAATCAATAAACCGTTTAATTCGTT

TCAGTTGAACTTTGGTCTCTGCGAAGGGCAATTCGTTTAAACCTGCAGGACTAGA

GGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGT

CACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGA

GGTTTGAACGCGCAGCCGCCAAGCCGAATTCTGCAGATATCACATGTCCTAGGAA

CTATCGATCCATCACACTGGCGGCCGCTCGACTAGAGCGGCCGCCACCGCGGTGG

AGCTCCAGCTTTTGCGGACCGAATCGGAAAGAACATGTGAGCAAAAGGCCAGCA AAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC

CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG

ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC

CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC

GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG

CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA

TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGG

CAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG

AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT

CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCC

GGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA

CGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA

CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAA

AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA

GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC

TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT

AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC

GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG

AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT

AATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACG

TTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA

TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCA

AAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC

AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCAT

CCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATA

GTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG

CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAA

AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC

ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAA

CAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA

ATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC

ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG By an "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV9 etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e. g., functional ITRs) of the virus. The ITRs need not be the wildtype nucleotide sequences, and may be altered, e. g, by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the CYP46A1 gene) and a transcriptional termination region.

The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5'and Y) with functional AAV ITR sequences. By "adeno-associated virus inverted terminal repeats " or "AAVITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e. g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc. Furthermore, 5'and 3'ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV 5, AAV6, etc. Furthermore, 5'and 3'ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

Particularly preferred are vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian CNS, particularly neurons. A review and comparison of transduction efficiencies of different serotypes is provided in Cearley CN et al., 2009. In one preferred example, AAV2 based vectors have been shown to direct long-term expression of transgenes in CNS, preferably transducing neurons. In other nonlimiting examples, preferred vectors include vectors derived from AAVrhlO serotype, which have also been shown to transduce cells of the CNS and particularly deep grey nucleus and diffusely cortex and white matter.

The selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo. Such control elements can comprise control sequences normally associated with the selected gene.

Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phophoglycerate kinase (PKG) promoter, CAG, neuronal promoters, promoter of Dopamine- 1 receptor and Dopamine-2 receptor, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e. g. , Stratagene (San Diego, CA). For purposes of the present invention, both heterologous promoters and other control elements, such as CNS-specific and inducible promoters, enhancers and the like, will be of particular use.

Examples of heterologous promoters include the CMV promoter. Examples of CNS specific promoters include those isolated from the genes from myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).

Examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia andaufin. The AAV expression vector which harbors the DNA molecule of interest bounded by AAV ITRs, can be constructed by directly inserting the selected sequence (s) into an AAV genome which has had the major AAV open reading frames("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e. g. , U. S. Patents Nos. 5,173, 414 and 5,139, 941; International Publications Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., 1988 ; Vincent et al., 1990; Carter, 1992 ; Muzyczka, 1992 ; Kotin, 1994; Shelling and Smith, 1994 ; and Zhou et al., 1994. Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5'and 3'of a selected nucleic acid construct that is present in another vector using standard ligation techniques. AAV vectors which contain ITRs have been described in, e. g. , U. S. Patent no. 5,139, 941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC") under Accession Numbers 53222,53223, 53224,53225 and 53226. Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5'and 3'of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells (notably neurons) can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e. g., Edge, 1981 ; Nambair et al., 1984 ; Jay et al., 1984. In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e. g. , Graham et al., 1973;, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981. Particularly suitable transfection methods include calcium phosphate coprecipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Feigner et al., 1987), and nucleic acid delivery using high- velocity microprojectiles (Klein et al., 1987).

For instance, a preferred viral vector, such as the AAVrhlO, comprises, in addition to a cholesterol 24-hydroxylase encoding nucleic acid sequence, the backbone of AAV vector with ITR derived from AAV-2, the promoter, such as the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus/p-actin hybrid promoter (CAG) consisting of the enhancer from the cytomegalovirus immediate gene, the promoter, splice donor and intron from the chicken P-actin gene, the splice acceptor from rabbit P-globin, or any neuronal promoter such as the promoter of Dopamine- 1 receptor or Dopamine-2 receptor with or without the wild-type or mutant form of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Delivery of the vectors

It is herein provided a method for treating Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, said method comprising:

(a) providing a vector as defined above, which comprises a NAGLU encoding nucleic acid; and

(b) delivering the vector intravenously.

In others words, the invention relates to a method for treating Sanfilippo syndrome type B (MPS3B) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprises the full sequence of N- acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject i intravenously.

The vectors used herein may be formulated in any suitable vehicle for delivery. For instance, they may be placed into a pharmaceutically acceptable suspension, solution or emulsion. Suitable mediums include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

A colloidal dispersion system may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

The preferred doses and regimen may be determined by a physician or base on study in NHP, dog and mouse model and also box study in NHP, and depend on the age, sex, weight, of the subject, and the stage of the disease. As an example, for delivery of NAGLU using a viral expression vector, each unit dosage of NAGLU expressing vector may comprise 1 pl to 500 ml of a composition including a viral expression vector in a pharmaceutically acceptable fluid at a concentration ranging from 10⁹ tolO¹⁷ viral genome per ml for example.

According to the invention, the vector of the invention may be delivered intravenously coupled with focused ultrasound to improve blood brain barrier crossing.

According to the invention, the ultrasounds may be administrated before, after or concomitantly with the administration of the AAV vector or the pharmaceutical composition, of the present invention.

In one embodiment, said ultrasounds have a frequency of about 100 to about 900 hHz, preferably of about 200 to about 500 hHz.

Pharmaceutical composition

A second object of the invention concerns a pharmaceutical composition for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof which comprises a therapeutically effective amount of a vector according to the invention.

According to the invention, the pharmaceutical composition is administrated to said subject intravenously.

By a "therapeutically effective amount" is meant a sufficient amount of the vector of the invention to treat Sanfilippo syndrome type B at a reasonable benefit/risk ratio applicable to any medical treatment.

It will be understood that the total daily dosage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range per adult per day. The therapeutically effective amount of the vector according to the invention that should be administered, as well as the dosage for the treatment of a pathological condition with the number of viral or non-viral particles and/or pharmaceutical compositions of the invention, will depend on numerous factors, including the age and condition of the patient, the severity of the disturbance or disorder, the method and frequency of administration and the particular peptide to be used.

The presentation of the pharmaceutical compositions that contain the vector according to the invention may be in any form that is suitable for intravenous administration.

In the pharmaceutical compositions of the present invention for intravenous, administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

A particular form useful according to this invention is DPBs + 0.001% pluronic.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The vector according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

Multiple doses can also be administered. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Body weight monitoring all along the study (A) and haematologic parameters

(B) and ionogram (C) follow up in the 2 NHP. Dotted line corresponds to the range of normal values when available.

Figure 2: NAGLU activity in several brain area of NHP (A), in other CNS, including cerebellar subcompartments, spinal cord, DRG and PNS structures (B) and in peripheral tissues

(C). For A: black correspond to IV only delivery of AAVPHP.eB, and grey to IV+IC of AAVPHP.eB-NAGLU and dashed to IV delivery of AAVMacPNSl -NAGLU; * white matter for IV only was not available. Dotted line corresponds to mean basal NAGLU activity in noninjected NHP.

Figure 3: (A) NaGlu activity in the brain, NaGlu activity was measured in tissue homogenates prepared from the mice brain hemispheres; (B) NaGlu activity in the cerebellum, and (C) Spinal cord. Data are means±SEM. ****p<0.001 were considered significant.

Figure 4: (A) Glycosaminoglycans (GAG) measurement was measured in tissue homogenates prepared from the mice brain hemispheres; (B) LAMP1 relative expression in the brain, protein extraction was obtained from cortical tissue extracts. Data are means±SEM. ***p<0.001 were considered significant.

EXAMPLES:

Material and Methods:

NHP experiments:

Adeno- Associated Viral Vector Construction and Production

AAV vectors were produced and purified by Atlantic Gene therapies (Translational Vector Core Research grade services, Nantes, France). AAVMacPNSl-CAG Naglu was produced by cloning the NAGLU sequence under the CAG promoter. The viral constructs for pAAVPHP.eB-CAG-hNAGLU contained the expression cassette consisting of the human NAGLU genes, driven by a CMV early enhancer/chicken b-actin (CAG) synthetic promoter surrounded by inverted terminal repeats (ITR) sequences of AAV2. Plasmid for AAVPHP.eB was obtained from Addgene (United States). The final titer of the batch was 1.7xl013vector genomes (vg)/ml.

Animal Model

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. NHP were housed in a temperature- and humidity-controlled animal facility (target temperature 20- 24°C, no specific humidity target in the guidelines (20 to 60%) with a 12 h light-dark cycle (no record for light). NHP diet pellets (ref 307, Safe) were given ad libitum except during the fasting experimental period and each day animal received one fruit or one vegetable and seeds or dry fruits. Tap water will be offered ad libitum in polycarbonate bottles.

Injection of AAV Vector

One male and one female non-human primates received lxl0E13vg total dose of AAVMacPNSl-CAG-NAGLU in the saphenous vein. Then, the monkeys were treated with corticoids for 8 days (from D-l to D7) following injection. The monkeys were followed-up for 6 weeks, with blood sampling at 1, 3 and 6 weeks. NAGLU activity was measured in various CNS and peripheral tissues.

A control animal that received AAVPHP.eB at the same concentration but with another transgene not related to NAGLU was used as a negative control for NAGLU activity to evaluate background expression.

Table 1: protocol of AAV injections Tissue Preparation

Animals were sacrificed by an intravenous administration of a lethal dose of Euthasol (140 mg/kg, Vetcare) 6 weeks after treatment. NHP was perfused intracardiacally with phosphate buffered saline (PBS), followed by 500mL of PFA 4%. Brain, spinal cord, sciatic nerve, heart, liver, gall gladder, lung, spleen and kidney were collected for analysis. Different structures of a cerebral hemisphere (frontal, temporal and occipital cortex, caudate, putamen, hippocampus, thalamus, corpus callosum, cerebellar white matter, pons) were dissected and frozen in liquid nitrogen. Spinal cord, sciatic nerve, dorsal root ganglia (DRG) heart, liver, lung, spleen and kidney were directly frozen in liquid nitrogen and stored in -80°C. For protein extraction from the same samples, tissue samples were crushed in liquid nitrogen and divided into two equals parts.

The brain was divided in 2 and one hemisphere was devoted to histological analysis and cryopreserved as well as a portion of spinal cord, sciatic nerve that were post-fixed overnight in 4% paraformaldehyde (PFA)ZPBSIX. Brain samples were rinsed three times in PBS IX and or frozen after PBS-sucrose gradients (5%, 10% and finally 20%) for 3-4 days each and cut into 40-um coronal section of brain. All other organs were embedded in paraffin and cut for transversal section of spinal cord or 5 um coronal section for the brain.

Protein Extraction and NAGLU Activity Quantification

Samples were homogenized in lysis buffer and NAGLU activity done as described previously.

Results:

NHP experiments

Preclinical study in non-human primates (NHP) The purpose of this NHP study was to perform a biodistribution, efficiency and first assessment of tolerance study in NHP after intravenous delivery of AAVMacPNSl-CAG-NaGlu at IE¹³ total dose with 2 animals. After dosing, animals were followed up for 6 weeks. All the study was run as a GLP-like study at the TIDU- GENOV with appropriate quality follow up. All NHP were followed for 6 weeks following surgery prior to necropsies.

IV was perfectly well tolerated in all animals, notably with no body weight change (Figure 1A) and normal blood formulation and ionogram all along the study even in all liver related parameters (Figure 2B and 2C). Importantly, NHP were monitored daily during the in-life period and none of them present any neurological symptoms or abnormalities, they all perfectly well tolerated the administration procedure.

Evaluation of a well know biomarker for neuronal integrity: NF-L is ongoing both in serum and CSF.

The second crucial point of our study was the evaluation of NAGLU activity in the brain and more largely in the whole CNS, the peripheral organs (Figure 2A, 2B and 2C).

A significant NAGLU activity was detected in brain regions, with a clear difference of IV delivery compared to baseline NAGLU activity in non-treated NHP demonstrating a significant increase in NAGLU expression notably in white matter (corpus callosum, general white matter, internal capsule) but also in caudate, putamen, thalamus (Figure 2A). To compare, previous results obtained by the group with IV and combine IV+IC delivery of AAVPHP.eB encoding NAGLU has been graphed to demonstrate the large superiority of IV AAVMacPNSl delivery that led to higher increase in NAGLU activity than combine IV+IC and thus demonstrate its potential for treating MPS3B disease.

For cerebellar regions, DRG, spinal cord and PNS, tend to superiority of AAVMacPNS 1 IV was also demonstrated compared to IV+IC AAVPHP.eB (Figure 2B). In peripheral organs, NAGLU activity was mostly in the range of non-injected controls except for kidney and spleen that tend to display higher Naglu activity.

At last, the inventors compare the level of NAGLU activity in serum and CSF in noninjected non-human primates versus injected non-human primate. They show a significant difference.

At last, the inventors demonstrate the tolerance pattern in the central nervous system by histology of the intravenously injection of the vector of the invention (AAVMacPNSl) compared to combining intracerebral and intravenous delivery of AAVPHP.eB. They show that the use of the vector of the invention is less invasive.

Indeed, intracerebral delivery of AAV is almost always associated to local reaction along the injection track (Piguet F et al. Human Molecular Genetics. 2010 Jan 1.19(1): 147-58 and Zerah M et al. P Hum Gene Ther Clin Dev. 2015 Jun; 26(2): 113-24) and this intravenous delivery led to more homogenous repartition in the central nervous system which allow important and homogenous bioavailability of the NAGLU enzyme to the cells diffusely in the brain.

Finally, the inventors begin to show that the use of the vector of the invention (AAVMacPNSl) intravenously allow to treat mouse model of the pathology (Ausseil J et al. PLoS One. 2008 and Vitry S. al. Am J Pathol. 2010) and dog model of the pathology. They begin to show that the vector is well tolerated and efficient.

Efficacy of AAVMacPNSl -NaGlu in MPSIIIB mouse model

Demonstration of the efficacy and therapeutic evaluation of AAVMacPNSl -NaGlu in the mouse model of the pathology was done and compared with AAVPHP.eB-NaGlu results. AAVPHP.eB-NaGlu and AAVMacPNSl -NaGlu were injected at a total dose of 5E1 Ivg in the tail vein of 4 weeks old MPSIIIB mice and their age-matched wild type (WT) littermates and necropsied at 4 weeks post-injection.

High NaGlu activity was measured in the brain, cerebellum, and spinal cord starting 4 weeks post-injection (Figure 3). Interestingly, the values obtained with AAVMacPNSl -NaGlu are similar to those obtained after treatment with AAVPHP.eB-NaGlu, and reduction of GAGs (Figure 4A) and LAMP1 (Figure 4B) are fully similar between both groups. Our data indicate that a single IV injection of AAVMacPNSl -NaGlu increases NaGlu activity in the CNS. Additional experiments are ongoing to study the effect of AAVMacPNSl -NaGlu on the neuropathological markers such as neuroinflammation.

As a conclusion, we clearly demonstrated a good efficacy of AAVMacPNSl to transduce mouse CNS after IV delivery and perfectly similar and reproducible therapeutic effect if we compare AAVPHP.eB and AAVmacPNSl in rodents validating the bridging study.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Heron B, Mikaeloff Y, Froissart R, Caridade G, Maire I, Caillaud C, et al. Incidence and Natural History of Mucopolysaccharidosis Type III in France and Comparison With United Kingdom and Greece. Am J Med Genet A (2011) 155A:58-68. doi: 10.1002/ajmg.a.33779. 2. Valstar MJ, Bruggenwirth HT, Olmer R, Wevers RA, Verheijen FW, Poorthuis BJ, et al. Mucopolysaccharidosis Type IIIB may Predominantly Present With an Attenuated Clinical Phenotype. J Inherit Metab Dis (2010) 33:759-67. doi: 10.1007/sl0545-010-9199-y.

3. Weber B, Guo XH, Kleijer WJ, van de Kamp JJ, Poorthuis BJ, Hopwood JJ. Sanfilippo Type B Syndrome (Mucopolysaccharidosis III B): Allelic Heterogeneity Corresponds to the Wide Spectrum of Clinical Phenotypes. Eur J Hum Genet (1999) 7:34-44. doi: 10.1038/sj.ejhg.5200242.

4. Beneto N, Vilageliu L, Grinberg D, Canals I. Sanfilippo Syndrome: Molecular Basis, Disease Models and Therapeutic Approaches. Int J Mol Sci (2020) 21 :7819-39. doi: 10.3390/ijms21217819.

5. Cheng SH. Gene Therapy for the Neurological Manifestations in Lysosomal Storage Disorders. J Lipid Res (2014) 55: 1827-38. doi: 10.1194/jlr.R047175.

6. Mij anovic O, Brankovic A, Borovjagin A, Butnaru DV, Bezrukov EA, Sukhanov RB, et al. Battling Neurodegenerative Diseases With Adeno- Associated Virus-Based Approaches. Viruses (2020) 12:460-85. doi: 10.3390/vl2040460.

7. Tardieu M, Zerah M, Husson B, de Bournonville S, Deiva K, Adamsbaum C, et al. Intracerebral Administration of Adeno-Associated Viral Vector Serotype rh.10 Carrying Human SGSH and SUMF1 cDNAs in Children With Mucopolysaccharidosis Type IIIA Disease: Results of a Phase I/II Trial. Hum Gene Ther (2014) 25:506-16. doi: 10.1089/hum.2013.238.

8. Cressant A, Desmaris N, Verot L, Brejot T, Froissart R, Vanier MT, et al. Improved Behavior and Neuropathology in the Mouse Model of Sanfilippo Type IIIB Disease After Adeno-Associated Virus-Mediated Gene Transfer in the Striatum. JNeurosci (2004) 24: 10229- 39. doi: 10.1523/JNEUROSCI.3558- 04.2004.

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Claims

CLAIMS:

1. A vector for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof, which vector comprises the full sequence of N-acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject intravenously.

2. The vector for use according to the claim 1, comprising a nucleic acid sequence that encodes the amino acid sequence SEQ ID N°2.

3. The vector for use according to any claims 1 to 2, comprising the nucleic acid sequence SEQ ID N°1 or a variant thereof.

4. The vector for use according to any claims 1 to 3, which is selected from the group of adenovirus, lentivirus, retrovirus, herpesvirus and Adeno-Associated Virus (AAV) vectors.

5. The vector for use according to any claims 1 to 4 which is an AAV vector.

6. The vector for use according to the claim 5, which is an AAVMacPNS 1.

7. A pharmaceutical composition for use in the treatment of Sanfilippo syndrome type B (MPS3B) in a subject in need thereof which comprises a therapeutically effective amount of a vector as defined in any one of claims 1 to 7.

8. A method for treating Sanfilippo syndrome type B (MPS3B) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a vector which comprises the full sequence of N-acetylglucosaminidase, alpha (NAGLU) encoding nucleic acid and wherein the vector is administrated to said subject i intravenously.

9. The vector for use according to the claims 1 to 6 or the method according to the claim 8, wherein a step of exposing the subject to ultrasounds is added.