ADENOASSOCIATED VIRUS VECTORS FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDERS
20170088859 · 2017-03-30
Inventors
- M Fàtima BOSCH TUBERT (Cerdanyola del Valles, ES)
- M Virginia HAURIGOT MENDOÇA (Barcelona, ES)
- Albert RIBERA SANCHEZ (Santa Eulàlia de Ronçana, ES)
Cpc classification
C12N2800/22
CHEMISTRY; METALLURGY
C12N2750/14143
CHEMISTRY; METALLURGY
A61P19/08
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
A61K38/47
HUMAN NECESSITIES
A61K48/005
HUMAN NECESSITIES
C12N9/2474
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
C12N2830/008
CHEMISTRY; METALLURGY
A61K48/0075
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
International classification
Abstract
The present invention provides new adenoassociated virus vectors and pharmaceutical compositions containing the same for the treatment of lysosomal storage disorders and specially, for the treatment of mucopolysaccharidoses Type IIIB.
Claims
1-19. (canceled)
20. A recombinant Adenoassociated Virus AAV9 vector comprising a CAG promoter comprising the nucleotide sequence of SEQ ID NO: 4, wherein the CAG promoter is operably linked to a nucleic acid encoding N-acetylglucosaminidase, alpha, wherein the N-acetylglucosaminidase, alpha comprises the amino acid sequence of SEQ ID NO: 1.
21. The recombinant AAV9 vector of claim 20, wherein the nucleic acid encoding N-acetylglucosaminidase, alpha exhibits at least 80% sequence identity with the nucleic acid comprising the sequence of SEQ ID NO: 2.
22. The recombinant AAV9 vector of claim 20, wherein the nucleic acid encoding N-acetylglucosaminidase, alpha exhibits at least 85% sequence identity with the nucleic acid comprising the sequence of SEQ ID NO: 3.
23. The recombinant AAV9 vector of claim 20, wherein the nucleic acid encoding N-acetyl alucosaminidase, alpha comprises the nucleic acid sequence of SEQ ID NO:2.
24. The recombinant AAV9 vector of claim 20, wherein the nucleic acid encoding N-acetylgiucosaminidase, alpha comprises the nucleic acid sequence of SEQ ID NO:3.
25. The recombinant AAV9 vector of claim 20, wherein the nucleic acid encoding N-acetylgiucosaminidase, alpha comprises the nucleic acid sequence of SEQ ID NO:19.
26. The recombinant AAV9 vector of claim 20, wherein the nucleic acid encoding N-acetylglucosaminidase, alpha comprises the nucleic acid sequence of SEQ ID NO:22.
27. A plasmid comprising a nucleic acid encoding N-acetylglucosaminidase, alpha, having the amino acid sequence of SEQ ID NO:1, wherein the nucleic acid encoding N-acetylglucosaminidase, alpha exhibits at least 80% sequence identity with SEQ ID NO: 2.
28. The plasmid of claim 27, wherein the plasmid is pAAV-CAG-cohNaglu, deposited under the terms of the Budapest Treaty under accession number DSM 26626, and comprises the nucleotide sequence of SEQ ID NO: 3 which encodes N-acetylglucosaminidase, alpha comprising the amino acid sequence of SEQ ID NO: 1.
29. The plasmid according to claim 27, wherein the plasmid is pAAV-CAG-hNaglu, deposited under the terms of the Budapest Treaty under accession number DSM 28568, and comprises the nucleotide sequence of SEQ ID NO: 2 which encodes N-acetylglucosaminidase, alpha comprising the amino acid sequence of SEQ ID NO: 1.
30. The plasmid according to claim 27, wherein the plasmid is pAAV-CAG-cohNaglu-version2, deposited under the terms of the Budapest Treaty under accession number DSM 32042, and comprises the nucleotide sequence of SEQ ID NO: 19 which encodes N-acetylglucosaminidase, alpha comprising the amino acid sequence of SEQ ID NO: 1.
31. The plasmid of claim 27, wherein the plasmid is pAAV-CAG-cohNaglu-version3, deposited under the terms of the Budapest Treaty under accession number DSM 32043, and comprises the nucleotide sequence of SEQ ID NO: 22 which encodes N-acetylglucosaminidase, alpha comprising the amino acid sequence of SEQ ID NO: 1.
32. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant AAV9 vector of claim 20.
33. The pharmaceutical composition of claim 32 which is in a form for intravenous or intracisternal administration.
34. A pharmaceutical composition comprising a therapeutically effective amount of the plasmid of claim 27.
35. The pharmaceutical composition of claim 34 which is in a form for intravenous or intracisternal administration.
36. A method for increasing the N-acetylgiucosaminidase, alpha activity in a living animal body, including a human, the method comprising administering to a subject in need thereof, the recombinant AAV9 vector of claim 20.
37. A method for increasing the N-acetylglucosaminidase, alpha activity in a living animal body, including a human, the method comprising administering to a subject in need thereof, the plasmid of claim 27.
38. A method for the treatment of mucopolysaccharidosis in a living animal body, including a human, the method comprising administering to a subject in need thereof, the recombinant AAV9 vector of claim 20.
39. The method of claim 38, wherein the mucopolysaccharidosis is mucopolysaccharidosis type IIIB.
40. A method for the treatment of mucopolysaccharidosis in a living animal body, including a human, the method comprising administering to a subject in need thereof, the plasmid of claim 27.
41. The method of claim 40, wherein the mucopolysaccharidosis is mucopolysaccharidosis type MB.
42. A method of producing the recombinant AAV9 vector of claim 20 comprising the steps of: i) providing a first vector comprising the sequence coding for the protein of interest interposed between a first Adenoassociated Virus (AAV) vector terminal repeat and a second AAV terminal repeat, a CAG promoter operably linked to a nucleic acid encoding the protein of interest; a second vector comprising an AAV rep gene and a AAV cap gene from AAV serotype 9; and a third vector comprising the adenovirus helper function gene; ii) co-transfecting competent cells with the vectors of step i); iii) culturing the transfected cells of step ii); and iv) purifying the expression vectors from the culture of step iii).
43. An isolated cell comprising N-acetylglucosaminidase, alpha comprising the amino acid sequence of SEQ ID NO:1, wherein the nucleic acid encoding the N-acetylgiucosaminidase, alpha exhibits at least 80% sequence identity with nucleic acid sequence of SEQ ID NO: 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DEPOSIT OF MICROORGANISMS
[0051] The plasmid pAAV-CAG-cohNaglu (SEQ ID NO: 6) was deposited on Nov. 13, 2012, under access number DSM 26626 at the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrae 7 B, D-38124 Braunschweig, Federal Republic of Germany.
[0052] The plasmid pAAV-CAG-hNaglu (SEQ ID NO: 5) was deposited on Mar. 13, 2014, under access number DSM 28568 at the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrae 7 B, D-38124 Braunschweig, Federal Republic of Germany.
[0053] The plasmid pAAV-CAG-cohNaglu-version2 (SEQ ID NO: 20) was deposited on Apr. 29, 2015, under access number DSM 32042 at the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrae 7 B, D-38124 Braunschweig, Federal Republic of Germany.
[0054] The plasmid pAAV-CAG-cohNaglu-version3 (SEQ ID NO: 23) was deposited on Apr. 29, 2015, under access number DSM 32043 at the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrae 7 B, D-38124 Braunschweig, Federal Republic of Germany.
DEFINITIONS
[0055] The term nucleotide sequence refers to a nucleic acid molecule, either DNA or RNA, containing deoxyribonucleotides or ribonucleotides respectively. The nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.
[0056] The term % sequence identity refers to the percentage of nucleotides of a candidate sequence that are identical to the nucleotides in the sequence of reference, after aligning the sequences to achieve the maximum % sequence identity. The % sequence identity can be determined by any methods or algorithms established in the art, such as the ALIGN, BLAST and BLAST 2.0 algorithms. See Altschul S, et al., Nuc Acids Res. 1977; 25:3389-3402 and Altschul S, et al., J Mol Biol. 1990; 215:403-410.
[0057] Herein, the % sequence identity is calculated dividing the number of nucleotides that are identical after aligning the sequence of reference and the candidate sequence, by the total number of nucleotides in the sequence of reference and multiplying the result by 100.
[0058] The terms codify or coding refer to the genetic code that determines how a nucleotide sequence is translated into a polypeptide or a protein. The order of the nucleotides in a sequence determines the order of amino acids along a polypeptide or a protein.
[0059] The term protein refers to a macromolecule composed of one or more linear chains of amino acids or polypeptides. Proteins can suffer post-translational modifications, like the conversion of a cysteine residue to 3-oxoalanine, glycosylation or metal binding. Glycosilation of a protein is the addition of different carbohydrates that are linked covalently to the amino acid chain.
[0060] The term effective amount refers to an amount of a substance sufficient to achieve the intended purpose. For example, an effective amount of an AAV9 vector to increase N-acetylglucosaminidase, alpha (Naglu) activity is an amount sufficient to reduce glycosaminoglycan accumulation. A therapeutically effective amount of an expression vector to treat a disease or disorder is an amount of the expression vector sufficient to reduce or eradicate the signs and symptoms of the disease or disorder. The effective amount of a given substance will vary with factors such as the nature of the substance, the route of administration, the size and species of the animal to receive the substance and the purpose of giving the substance. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
[0061] The term individual refers to a mammal, preferably human or non-human mammal, more preferably mouse, rat, other rodents, rabbit, dog, cat, pig, cow, horse or primate, further more preferably human.
[0062] The term operably linked refers to the functional relation and the location of the promoter sequence with respect to the gene of interest (e.g. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence). Generally, a promoter operably linked is contiguous to the sequence of interest. However, an enhancer does not have to be contiguous to the sequence of interest to control its expression.
[0063] The term tropism refers to the way in which different viruses have evolved to preferentially target specific host species, or specific cell types within those species.
[0064] The term gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product (e.g. a protein polypeptide, peptide or functional RNA) whose production in vivo is desired. For example, the genetic material of interest can encode an enzyme, hormone, receptor, or polypeptide of therapeutic value.
[0065] The terms recombinant viral vector, viral vector, recombinant vector or vector refer to an agent obtained from a naturally-occurring virus through genetic engineering techniques capable of transferring genetic material (e.g. DNA or RNA) of interest to a cell, which results in production of the product encoded by that said genetic material (e.g. a protein polypeptide, peptide or functional RNA) in the target cell. In the context of the present invention, a recombinant vector or a vector is to be understood as being a capsid protein as well as the genetic material contained within used to transfer said genetic material into a cell. By referring to a recombinant vector or to a vector through a nucleotide sequence, it means that it refers to a recombinant vector or a vector whose genome is as set forth in the corresponding sequence listing (SEQ ID).
[0066] The term recombinant plasmid or plasmid refers to a small, circular, double-stranded, self-replicating DNA molecule obtained through genetic engineering techniques capable of transferring genetic material of interest to a cell, which results in production of the product encoded by that said genetic material (e.g. a protein polypeptide, peptide or functional RNA) in the target cell. Furthermore, the term recombinant plasmid or plasmid also refers to a small, circular, double-stranded, self-replicating DNA molecule obtained through genetic engineering techniques used during the manufacturing of viral vectors as carriers of the recombinant vector genome.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The present invention provides new recombinant vectors for the treatment of diseases, in particular for the treatment of mucopolysaccharidoses type III (MPSIII), especially MPSIIIB
[0068] Apart from the genetic material, the recombinant vector may also contain different functional elements that include control elements for transcription like promoters or operators, transcription factors binding regions or enhancers and control elements for the initiation or termination of translation.
[0069] The vectors according to the invention are adenoassociated vectors (AAV) that are used to transfer the gene of interest. They have proved to have a high efficiency in transducing post-mitotic cells in wide range of tissue. In the context of the present invention, the vectors are used to deliver the human N-acetylglucosaminidase, alpha (hNaglu) polynucleotide (SEQ ID NO: 2) or a codon optimized human N-acetylglucosaminidase, alpha (cohNaglu) polynucleotide (SEQ ID NO: 3). An adenoassociated vector is a vector derived from the genome of an adenoassociated virus of the family of parvoviridae. The adenoassociated virus genome is built of single-stranded deoxyribonucleic acid (ssDNA). These vectors infect mammals but are non-pathogenic (i.e. do not cause disease). They can infect dividing or non-dividing cells, and their tropism changes depending on the serotype. The serotype is the classification of the viruses groups, depending on their capsid antigens. The serotype of adenoassciated virus, determined by its capsid protein, defines the virus tropism and allows its entry into a specific cell type. In the context of the present invention, the serotype 9 of the adenoassociated virus vectors (AAV9) shows the best ability to deliver the genetic material to the brain as well as to peripheral organs upon a single administration.
[0070] The inventors have surprisingly found that the association, in the same entity, of the AAV9 capsid with a nucleotide sequence coding for the N-acetylglucosaminidase, alpha, together with a specific CAG promoter allows a long-lasting expression of the missing enzyme in all areas of the brain. As a consequence the lysosomal accumulation of glycosaminoglycan (GAG) is corrected, preventing by that way the neurological alterations characteristic of the MSPIII diseases, and in particular of the MPSIIIB This effect has been obtained even in the olfactory bulb, which is distant form the point of administration of the vectors. Further the AAV9 vectors according to the invention delivered into the cerebrospinal fluid were able to reach the systemic circulation to transduce the liver. The production and secretion of the enzyme by liver cells resulted in an increase of N-acetylglucosaminidase, alpha activity in serum, ultimately leading to the reduction of lysosomal pathology in many somatic tissues. This represents a clear advantage of the vectors according to the invention over the existing approaches that only partially corrected the clinical signs of the disease and usually exert their effect either in the brain or in the systemic circulation, but not in both.
[0071] Accordingly the present invention relates to recombinant AAV9 vectors containing a CAG promoter (SEQ ID NO: 4) linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
[0072] In particular, the recombinant AAV9 vectors of the present invention contain a CAG promoter (SEQ ID NO: 4) linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 80% sequence identity to SEQ ID NO: 2. More preferably, the AAV9 recombinant vectors of the present invention contain a CAG promoter (SEQ ID NO: 4) linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 84% sequence identity to SEQ ID NO: 2. In particular the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has a 84%, 87%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2.
[0073] In another particular embodiment the recombinant AAV9 vectors of the present invention contains a CAG promoter (SEQ ID NO: 4) linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 85% sequence identity to SEQ ID NO: 3. In particular the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has a 85%, 87%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3.
[0074] In a preferred embodiment of the invention, the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 2.
[0075] In a further preferred embodiment of the invention, the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 3. The sequence as set forth in SEQ ID NO: 3 presents 84% sequence identity with SEQ ID NO: 2.
[0076] In a still further preferred embodiment of the invention, the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 19.
[0077] In a still further preferred embodiment of the invention, the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 22.
[0078] The AAV9 vectors according to the present invention contain a promoter that control the translation and transcription of the gene of interest. A promoter is a nucleotide sequence operably linked to said gene of interest. The promoter used in the present invention is the CAG promoter which refers to the combination comprising the cytomegalovirus early enhancer element and the chicken -actin promoter. It further includes a portion of -globin intron that confers stability to the m RNA derived from the gene of interest, See Alexopoulou A, et al., BMC Cell Biology 2008; 9(2): 1-11. The CAG promoter included in the AAV9 vectors of the present invention has a sequence SEQ ID NO: 4. In particular this CAG promoter proved to be more efficient than the CMV promoter usually used in the art.
[0079] In a further preferred embodiment of the invention, the recombinant AAV9 vector is chosen from AAV9-CAG-hNaglu (SEQ ID NO: 9), AAV9-CAG-cohNaglu (SEQ ID NO: 10) and AAV9-CAG-cohNaglu-version2 (SEQ ID NO: 21) and AAV9-CAG-cohNaglu-version3 (SEQ ID NO: 24). Preferably, the recombinant AAV9 vector is chosen from AAV9-CAG-hNaglu (SEQ ID NO: 9), AAV9-CAG-cohNaglu (SEQ ID NO: 10). More specifically, the recombinant AAV9 vectors of the present invention are composed of the viral capsid of the serotype 9 of human adenoassociated virus and a modified genome containing the Inverted Terminal Repeats (ITRs) of human adenoassociated virus serotype 2, the CAG promoter, the Coding Sequence (CDS) of the human alpha N-acetylglucosamidinase (Naglu) gene and the polyA from the rabbit beta-globin gene.
[0080] The present invention also related to plasmids that contain the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1. In particular the plasmids according to the present invention contain a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 80% sequence identity to SEQ ID NO: 2. Preferably, the plasmids according to the present invention contain a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 84% sequence identity to SEQ ID NO: 2. In particular the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has a 84%, 87%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2.
[0081] In a preferred embodiment, the plasmids according to the present invention contain a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 85% sequence identity to SEQ ID NO: 3. In particular the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has a 85%, 87%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3.
[0082] These plasmids are useful to produce the recombinant AAV9 vectors of the present invention by transfection of HEK293 cells using methods known in the state of the art.
[0083] In a preferred embodiment of the invention, the nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 2.
[0084] In another preferred embodiment of the invention, the nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 3.
[0085] In another preferred embodiment of the invention, the nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 19.
[0086] In another preferred embodiment of the invention, the nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 22.
[0087] In a more preferred embodiment, the plasmids of the invention are chosen from pAAV-CAG-hNaglu (SEQ ID NO: 5), pAAV-CAG-cohNaglu (SEQ ID NO: 6), pAAV-CAG-cohNaglu-version2 (SEQ ID NO: 20) and pAAV-CAG-cohNaglu-version3 (SEQ ID NO: 23), and especially chosen from pAAV-CAG-hNaglu (SEQ ID NO: 5) and pAAV-CAG-cohNaglu (SEQ ID NO: 6), and preferably the plasmid is pAAV-CAG-cohNaglu (SEQ ID NO: 6).
[0088] The present invention further provides a method for the production of the adenoassociated viral recombinant vectors AAV9 according to the invention. The process comprises the steps of: [0089] i) providing a first vector comprising the sequence coding for the protein of interest interposed between a first AAV terminal repeat and a second AAV terminal repeat, a CAG promoter operably linked to the sequence coding for the protein of interest; a second vector comprising an AAV rep gene and a AAV cap gene from serotype 9; and a third vector comprising the adenovirus helper function gene; [0090] ii) co-transfecting competent cells with the vectors of step i); [0091] iii) culturing the transfected cells of step ii); and [0092] iv) purifying the expression vectors from the culture of step iii).
[0093] In a preferred embodiment, the AAV first and second terminal repeats of the first vector are ITRs from the AAV serotype 2. In another preferred embodiment, the AAV rep genes of the second vector are from the AAV serotype 2. In another preferred embodiment, the competent cells are HEK293 cells.
[0094] The invention also provides a method for the preparation of the plasmids according to the invention, comprising the step of: [0095] i) excising the sequence coding for the protein of interest from the starting plasmid, by digestion, in particular using MIuI/EcoRI, [0096] ii) cloning the sequence coding for the protein of interest between two restriction sites of the AAV backbone plasmid pAAV-CAG, hereby obtaining the corresponding plasmid including the sequence coding for the protein of interest.
[0097] The present invention contemplates, in an additional aspect, pharmaceutical compositions containing a therapeutically effective amount of the AAV9 vectors described therein, or a therapeutically effective amount of the plasmids described therein.
[0098] Pharmaceutical compositions of the invention comprise the recombinant AAV9 vectors in a pharmaceutically acceptable carrier. The composition may also comprise at least one auxiliary substance. The auxiliary substances can be selected among carriers, excipients, solvents, diluents, or adjuvants. Acceptable carriers, diluent or adjuvants are non-toxic and are preferably inert at the dosage and concentrations employed and include buffers such as phosphate, citrate or other organic acids; antioxidants; low molecular weight polypeptides, proteins such as serum albumin, gelatin or immunoglobulins; hydriophilic polymers; aminoacids; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents; sugar alcohols such as mannitol or sorbitol, salt forming couterions such as sodium; and/or non-ionic surfactants such as polyethylene-polyoxypropylene block copolymer (Pluronic F68) polyethylene glycol (PEG).
[0099] In a preferred embodiment, the pharmaceutical compositions according to the invention are suitable for parenteral administration. Examples of parenteral administration are intravenous, subcutaneous, intracisternal and intramuscular injections. Preferably, the pharmaceutical composition according to the invention is suitable for intravenous or intracisternal administration. Compositions suitable for such parenteral administration include sterile aqueous solutions or dispersions, sterile powders for extemporaneous preparation of sterile solutions or dispersions. Advantageously the pharmaceutical compositions according to the invention are preserved from contaminating action of bacteria and fungi.
[0100] The daily dosage for humans and animals may vary depending on factors that have their basis in the respective species or other factors, such as age, sex, weight or degree of illness and so forth.
[0101] Another aspect of the present invention relates to the therapeutical use of the AAV9 vectors described hereinbefore, or the plasmids described hereinbefore. As mentioned above the recombinant AAV9 vectors according to the invention achieve an expression of the missing Naglu enzyme, thus correcting the lysosomal accumulation of GAGs. This allows correcting all clinical signs of the mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB In this respect the present invention also concern the recombinant AAV9 vectors described hereinbefore, or the plasmids described hereinbefore for use as a medicament.
[0102] In particular, the invention relates to the recombinant AAV9 vectors described hereinbefore, or the plasmids described hereinbefore for increasing the alpha N-glucosaminidase activity in the body.
[0103] In a further preferred aspect, the present invention relates to the recombinant AAV9 vectors described hereinbefore, or the plasmids described hereinbefore for treatment of mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB
[0104] In a still further embodiment, the present invention relates to the use of the recombinant AAV9 vectors described hereinbefore, or the plasmids described hereinbefore for the manufacture of a medicament useful for the treatment of mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB
[0105] Another embodiment of the present invention is directed to the method of treatment of mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB, comprising the step of administering at least a recombinant AAV9 vector described hereinbefore, or at least a plasmid described hereinbefore to a subject in need thereof.
[0106] The present invention further provides an isolated cell comprising the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1. In particular the cell according to the invention comprises a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 80% sequence identity to SEQ ID NO: 2. Preferably, the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 84% sequence identity to SEQ ID NO: 2. In particular the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has a 84%, 87%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2. In a further particular aspect, the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has at least 85% sequence identity to SEQ ID NO: 3, and preferably 85%, 87%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3.
[0107] In a preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 2 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
[0108] In another preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 3 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
[0109] In another preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 19 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
[0110] In another preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 22 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
[0111] The following examples are merely illustrative of certain embodiments of the invention and cannot be considered as restricting it in any way.
General Procedures
[0112] 1. Recombinant AAV Vectors
[0113] The AAV vectors described herein were obtained by triple transfection. The materials required for making the vectors were: HEK293 cells (expressing E1 genes), helper plasmid providing adenovirus function, plasmid providing AAV rep genes from serotype 2 and cap genes from the serotype 9 (AAV9) and, finally, the backbone plasmid with AAV2 ITRs and the construct of interest.
[0114] To generate N-acetylglucosaminidase, alpha-expressing AAV vectors, the non-optimized or optimized CDS of human, murine or canine N-acetylglucosaminidase, alpha were cloned into an AAV backbone plasmid under the control of the ubiquitous hybrid CAG promoter.
[0115] Vectors were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita T, et al., Gene Ther. 1998; 5:938-945 and Wright J, et al., Mol. Ther. 2005; 12:171-178. Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, N.Y., US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by the viral ITRs of serotype 2 AAV (described above); 2) a plasmid carrying the AAV rep2 and the cap9 genes; and 3) a plasmid carrying the adenovirus helper functions. Vectors were purified by two consecutives cesium chloride gradients using an optimized protocol as previously described. See Ayuso E, et al., Gene Ther. 2010; 17:503-510. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at 80 C. until use.
[0116] The vectors of the present invention were constructed according to molecular biology techniques well known in the art.
[0117] 2. Animals
[0118] A congenic mutant C57Bl/6J N-acetylglucosaminidase, alpha-deficient mouse (MPSIIIB) model was purchased from The Jackson Laboratory (Bar Harbor, Me., USA. Stock 003827). See Li et al., Proc Natl Acad Sci. 1999; 96(25):14505-10. Affected MPSIIIB and healthy control mice were inbred from heterozygous founders. Genotype was determined on genomic DNA from tail-clipped samples with a PCR analysis that amplifies a sequence encompassing the targeted mutation. The sequences of the respective sense and antisense primers were: Forward Primer: 5-GTC GTC TCC TGG TTC TGG AC-3 (SEQ ID NO: 13), Reverse Primer: 5-ACC ACT TCA TTC TGG CCA AT-3 (SEQ ID NO: 14), Reverse Primer Mutation: 5-CGC TTT CTG GGC TCA GAG-3 (SEQ ID NO: 15). Mice were fed ad libitum with a standard diet (Harlan, Tekland)) and maintained under a light-dark cycle of 12 h (lights on at 9:00 A.M.).
[0119] 3. Hydrodynamic Delivery of hNAGLU-Encoding Plasmids to Mice
[0120] For hydrodynamic delivery of pAAV-CAG-hNaglu, pAAV-CAG-cohNaglu-version2 and pAAV-CAG-cohNaglu-version3 plasmids, 2-month-old MPSIIIB and wild-type animals received through tail vein injection in <5 seconds a total dose of 50 g of plasmid in a volume equal to 10% of the body weight of the animal. This technique results in expression of plasmid-encoded transgenes mainly in the liver. See Liu et al., Gene Ther. 1990; 6(7):1258-66. As control, a cohort of mice received and equal volume of saline solution. Mice were sacrificed either at 1 week after hydrodynamic injection of the plasmids. Organs were harvested as described in the following section.
[0121] 4. Vector Administration and Sample Collection
[0122] For intracisternal delivery of AAV9-CAG-comNaglu vectors to mice, a total dose of 310.sup.10 vg were injected to the cisterna magna of 2-month-old MPSIIIB animals. A similar cohort of animals was injected with 3.910.sup.10 vg control non-coding (AAV9-null) vector. At 5 months of age, i.e. 3 months post vector administration, mice were anesthetized and then transcardially perfused with 10 ml of PBS to completely clear blood from tissues. The entire brain and multiple somatic tissues (including liver, spleen, pancreas, kidney, lung, heart, skeletal muscle and testicles) were collected and either frozen in liquid nitrogen and stored at 80 C. or immersed in formalin for subsequent histological analyses.
[0123] For intravenous delivery of AAV9-CAG-hNaglu and AAV9-CAG-cohNaglu vectors, 2-month-old MPSIIIB animals received a total dose of 510.sup.11 vg through tail vein injection. At 4 months of age, i.e. 2 months post vector administration, mice were sacrificed and organs were harvested as described in the previous paragraph.
[0124] For intracisternal delivery of AAV9-CAG-hNaglu and AAV9-CAG-cohNaglu vectors, a total dose of 9.310.sup.9 vg were injected in the cisterna magna of 2-month-old MPSIIIB animals. At 4 months of age, i.e. 2 months post vector administration, mice were sacrificed and organs were harvested as described in the previous paragraph.
[0125] For intracisternal delivery of AAV9-CAG-cocNaglu vectors to dogs, a total dose of 6.510.sup.12 vg was administered to healthy adult Beagle dogs via cisterna magna injection. Two of the animals received an intravenous injection of 110.sup.11 vg/kg of AAV9-null vectors 6 weeks prior to administration of Naglu vectors to pre-immunize them against AAV9. First weekly, and then monthly, CSF and serum samples were collected and stored at 80 C.
[0126] 5. Quantification of Vector Genome Copy Number
[0127] Tissues (100 mg) were digested overnight (ON) at 56 C. in 400 l of Proteinase K solution (0.2 mg/ml). Total DNA was isolated from supernatants by extraction using standard techniques. DNA was resuspended in distilled water and quantified using a NanoDrop ND-1000 (NanoDrop, Wilmington, Del., USA). Vector genome copy number in 20 ng of total DNA was determined by quantitative real time PCR with primers and probe specifics for the murine N-acetylgluosaminidase, alpha transgene that do not amplify the endogenous genomic locus. Forward primer: 5-GCC GAG GCC CAG TTC TAC-3 (SEQ ID NO: 16); Reverse primer: 5-TTG GCG TAG TCC AGG ATG TTG-3 (SEQ ID NO: 17); Probe: 5-AGC AGA ACA GCA GAT ACC AGA TCA CCC-3 (SEQ ID NO: 18). The final values were determined by comparing to a reference standard curve, built from serial dilutions of the linearized plasmid used for AAV vector production spiked into 20 ng non-transduced genomic DNA.
[0128] 6. N-Acetylglucosaminidase, Alpha Activity and Glycosaminoglycan Quantification
[0129] Liver and brain samples were sonicated in Mili-Q water. Serum was analysed unprocessed. N-acetylglucosaminidase, alpha activity was determined with a 4-methylumbelliferone-derived fluorogenic substrate (Moscerdam Substrates, Oegstgeest, NL), as described previously. See Marsh and Fensom, Clin Genet. 1985; 27(3):258-262. Brain and liver activity levels were normalized against the total amount of protein, quantified using Bradford protein assay (Bio-Rad, Hercules, Calif., US). Serum activity was normalized against volume.
[0130] For glycosaminoglycan (GAG) quantification, tissue samples were weighted and then digested with proteinase K and extracts were clarified by centrifugation and filtration. GAG levels were determined in tissue extracts with the Blyscan sulfated glycosaminoglycan kit (Biocolor, Carrickfergus, County Antrim, GB), using chondroitin 4-sulfate as standard. The levels of GAG were normalized to wet tissue weight.
[0131] 7. Activity of Other Lysosomal Enzymes
[0132] IDUA activity was measured in 15 g of protein incubated for 1 h at 37 C. with 4-methylumbelliferyl -N-iduronide (Glycosynth). For IDS activity, 15 g of protein were first incubated with 4-methylumbelliferyl--L-iduronide-2-sulphate (Moscerdam Substrates) for 4 h at 37 C., followed by a second 24 h incubation at 37 C. with a pool of lysosomal enzymes from bovine testis (LEBT-M2, Moscerdam Substrates). SGSH activity was measured as previously described. See Haurigot et al., J Clin Invest 2013; 123(8):3254-71. For GUSB activity, 10 g of protein were incubated with 4-methylumbelliferyl--D-glucuronide (Sigma) at 37 C. for 1 h. HEXB activity was assayed by incubation of 0.1 g of protein with 4-methylumbelliferyl N-acetyl--D-glucoaminide (Sigma) for 1 h at 37 C. After stopping reactions by increasing the pH, released fluorescence was measured with FLx800 fluorimeter (BioTek Instruments). All enzyme activities were normalized against total protein content quantified by Bradford (Bio-Rad).
[0133] 8. Histological Analyses
[0134] Tissues were fixed for 12-24 h in formalin, embedded in paraffin and sectioned. For immunohistochemical detection of LAMP1 in somatic tissues, paraffin sections were subjected to heat-induced epitope retrieval in citrate buffer, pH 6, and then incubated overnight at 4 C. with rat anti-LAMP1 antibody (1D4B; Santa Cruz Biotechnology, Santa Cruz, Calif., US) diluted at 1:100 and subsequently incubated with biotinylated rabbit anti-rat antibody (Dako, Glostrup, DK) at 1:300. For immunohistochemical detection of LIMP2 in the brain, paraffin sections were incubated overnight at 4 C. with rabbit anti-LIMP2 antibody (NB400; Novus Biologicals, Littleton, Colo., USA) diluted at 1:100 and subsequently incubated with biotinylated goat anti-rabbit antibody (31820; Vector Laboratories, Burlingame, Calif., USA) at 1:300. For GFAP immunostaining in brain samples, paraffin sections were incubated overnight at 4 C. with rabbit anti-GFAP antibody (Ab6673; Abcam, Cambridge, UK) diluted at 1:1000 and subsequently incubated with biotinylated goat anti-rabbit antibody (31820; Vector Laboratories, Burlingame, Calif., USA) at 1:300. LAMP1, LIMP2 and GFAP signals were amplified by incubating sections with ABC-Peroxidase staining kit (Thermo Scientific, Waltham, Mass., US) at 1:100 dilution and visualized using 3,3-diaminobenzidine (Sigma-Aldrich, St. Louis, Mo., US) as a chromogen. Brightfield images were obtained with an optical microscope (Eclipse 90i; Nikon, Tokyo, JP).
[0135] To stain microglial cells in brain samples, paraffin sections were incubated overnight at 4 C. with Bsi-B4 lectin (L5391; Sigma-Aldrich, St. Louis, Mo., USA) diluted at 1:100. Bsi-B4 signal was visualized using 3,3-diaminobenzidine (Sigma-Aldrich, St. Louis, Mo., US) as a chromogen. Brightfield images were obtained with an optical microscope (Eclipse 90i; Nikon, Tokyo, JP).
[0136] The NIS Elements Advanced Research 2.20 software was used to quantify LIMP2, GFAP, and Bsi-B4 signals in 3-5 images of each brain region (original magnification, 20) per animal, using the same signal threshold settings for all animals. Then, the percentage of positive area was calculated, i.e., the area, in pixels, with a positive signal over the total tissue area in the image.
[0137] 9. Transmission Electron Microscopic Analysis
[0138] Mice were sacrificed by an overdose of isofluorane (Isofluo, Labs. Esteve, Barcelona, ES) and perfused via inferior vena cava with 1 ml of 2.5% glutaraldehyde and 2% paraformaldehyde. A small portion (approximately 1 mm.sup.3) of the lateral lobe of the liver and of the cerebral cortex were sectioned and incubated for 2 hours at 4 C. in the same fixative. After washing in cold cacodylate buffer, the specimens were postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, and then dehydrated through a graded ethanol series and embedded in epoxy resin. Ultrathin sections (600-800 ) from the resin blocks were stained using lead citrate and examined in a transmission electron microscope (H-7000; Hitachi, Tokyo, JP).
[0139] 10. Transcriptomic Analysis
[0140] Half mouse brain (250 mg) was mechanically homogenized and total RNA was isolated with mirVana (Ambion, Life Technolo-gies). cDNA was synthesized and subsequently hybridized in the GeneChip Mouse Gene 2.1 ST 16 array plate (Affymetrix) by Progenika Biopharma (Spain); sample processing, hybridization and scanning were carried out following Affymetrix recommended protocols and equipment. Data normalization was done by RMA (Robust Multiarray averaging) method using Affymetrix Expression Console tool, obtaining log 2 transformed normalized values. Data were filtered to focus the analysis on known coding sequences, obtaining an initial list of 26688 altered genes, which were subsequently refiltered to remove genes with variance below the 75th percentile. This process generated a working list of 6672 genes. For differentially expressed genes, FDR (False Discovery Rate) criteria <0.1 with 80% confidence was established. For clustering analysis, data were standardized and represented as heatmap using the J-Express Pro software (jexpress.bioinfo.no). Functional analysis was performed using Genecodis Tool 2.0 (genecodis2.dacya.ucm.es). Array data have been submitted to ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/; accession code: E-MTAB-2984).
[0141] 11. Behavioural Assessment
[0142] Behavioural changes were assessed through the Open-field test. Animals were placed in the lower left corner of a brightly lit chamber (414130 cm). The surface of the arena was divided in three concentric squares: centre (1414 cm), periphery (2727 cm) and border (4141 cm). Exploratory behaviour and general activity were recorded during the first two minutes using a video-tracking system (Smart Junior, Panlab). The test was always performed at the same time of day (9:00 am to 1:00 pm) to minimize influence of circadian cycles.
[0143] 12. Statistical Analysis
[0144] All results are expressed as meanSEM. Statistical comparisons were made using one-way ANOVA, and multiple comparisons between control and treatment groups will be made using Dunnett's post test. Statistical significance was considered if P<0.05. The Kaplan-Meier method will be used to analyze survival, and the log-rank test for comparisons.
EXAMPLES
Example 1: Construction of pAAV-CAG-hNaglu
[0145] The human N-acetylglucosaminidase, alpha coding sequence (CDS) was utilized as starting material (NCBI Reference Sequence: NM_000263) and chemically synthesized for this purpose (GeneArt; Life Technologies). The CDS was received cloned inside the plasmid pMA (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3 ends, respectively. N-acetylglucosaminidase, alpha CDS was excised by MluI/EcoRI digestion and then cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG (AmpR). The resulting plasmid was named pAAV-CAG-hNaglu (accession number DSM 28568). See SEQ ID NO: 5, and
[0146] The pAAV-CAG plasmid had been previously generated and contained the ITRs from the AAV2 genome, the CAG promoter, and the polyA signal from rabbit -globin, as well as a multicloning site for cloning of CDSs of interest. The CAG promoter is a hybrid promoter composed of the CMV early/intermediate enhancer and the chicken -actin promoter. This promoter is able to drive a potent expression ubiquitously. See Sawicki J et al., Exper Cell Res. 1998; 244:367-369, Huang J et al., J Gene Med. 2003; 5:900-908, Liu Y et al., Exp Mol Med. 2007; 39(2):170-175.
Example 2: Production of AAV9-CAG-hNaglu
[0147] Vectors AAV9-CAG-hNaglu (SEQ ID NO: 9 and
Example 3: Construction of pAAV-CAG-cohNaglu
[0148] Expression cassettes including an optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu) were designed and obtained. The sequence optimization (GeneArt) was performed to maximize the efficiency of N-acetylglucosaminidase, alpha protein production in human beings through elimination of cryptic splice sites and RNA destabilizing sequence elements for increased RNA stability, addition of RNA stabilizing sequence elements, codon optimization and G/C content adaptation, avoidance of stable RNA secondary structures amongst others changes. The optimized CDS was received cloned in the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0149] The pMA-RQ-cohNaglu plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu plasmid (accession number DSM 26626). See SEQ ID NO:6 and
Example 4: Production of AAV9-CAG-cohNaglu
[0150] Vectors AAV9-CAG-cohNaglu (SEQ ID NO: 10 and
Example 5: Construction of pAAV-CAG-cohNaglu-Version2
[0151] Expression cassettes including a second optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu-version2) were designed and obtained. The optimized CDS (DNA 2.0) was received cloned in the plasmid pJ208 (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0152] The pJ208-cohNaglu-version2 plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha-version2 CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu-version2 plasmid (accession number DSM 32042). See SEQ ID NO:20 and
Example 6: Production of AAV9-CAG-cohNaglu-version2
[0153] Vectors AAV9-CAG-cohNaglu-version2 (SEQ ID NO: 21 and
Example 7: Construction of pAAV-CAG-cohNaglu-version3
[0154] Expression cassettes including a third optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu-version3) were designed and obtained. The optimized CDS (GenScript, Inc) was received cloned in the plasmid pUC57 (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0155] The pUC57-cohNaglu-version3 plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha-version3 CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu-version3 plasmid (accession number DSM 32043). See SEQ ID NO:23 and
Example 8: Production of AAV9-CAG-cohNaglu-version3
[0156] Vectors AAV9-CAG-cohNaglu-version3 (SEQ ID NO: 24 and
Example 9: Construction of pAAV-CAG-comNaglu
[0157] The CDS for murine N-acetylglucosaminidase, alpha (NCBI Reference Sequence: NM_013792) was subjected to sequence optimization (GeneArt; Life Technologies). The optimized CDS was received cloned inside the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0158] The MluI/EcoRI optimized murine N-acetylglucosaminidase, alpha CDS fragment was excised from the pMA-RQ plasmid and subsequently cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG. The resulting plasmid was named pAAV-CAG-comNaglu. See SEQ ID NO: 7 and
Example 10: Production of AAV9-CAG-comNaglu
[0159] Vectors AAV9-CAG-comNaglu (SEQ ID NO: 11 and
Example 11: Construction of pAAV-CAG-cocNaglu
[0160] The CDS for canine N-acetylglucosaminidase, alpha (NCBI Reference Sequence: XM_548088.4) was subjected to sequence optimization (GeneArt; Life Technologies). The optimized CDS was received cloned inside the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0161] The MluI/EcoRI optimized canine N-acetylglucosaminidase, alpha CDS fragment was excised from the pMA-RQ plasmid and subsequently cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG. The resulting plasmid was named pAAV-CAG-cocNaglu. See SEQ ID NO: 8 and
Example 12: Production of AAV9-CAG-cocNaglu
[0162] Vectors AAV9-CAG-cocNaglu (SEQ ID NO: 12 and
Example 13: Hydrodynamic Delivery of the Plasmid pAAV9-CAG-hNaglu
[0163] A total dose of 50 g of the plasmid pAAV9-CAG-hNaglu containing the wild-type human N-acetylglucosaminidase, alpha expressing cassette were administered to 2-month-old MPSIIIB mice via hydrodynamic tail vein (HDTV) injection. This technique targets expression of the delivered plasmid to the liver. See Liu et al., Gene Ther. 1990; 6(7):1258-66.
[0164] One week post plasmid delivery, a considerable increase in N-acetylglucosaminidase, alpha activity over pre-treatment levels was documented in the liver and serum of all the animals administered with wild-type human N-acetylglucosaminidase, alpha-coding plasmids. See
Example 14: Intravenous Delivery of AAV9-CAG-hNaglu
[0165] A total dose of 510.sup.11 vector genomes of AAV9-CAG-hNaglu vectors was administered to 2-month-old MPSIIIB mice via tail vein injection.
[0166] Consistent with the high tropism of AAV9 vectors for the liver, two months after administration treated animals showed high levels of N-acetylglucosaminidase, alpha activity in this organ (700% of the activity levels observed in healthy animals), which completely eliminated or considerably reduced the pathological accumulation of GAGs observed in the somatic tissues of untreated MPSIIIB mice. See
Example 15: Intravenous Delivery of AAV9-CAG-cohNaglu
[0167] A total dose of 510.sup.11 vector genomes of AAV9-CAG-cohNaglu vectors was administered to 2-month-old MPSIIIB mice via tail vein injection.
[0168] Two months after administration, treated animals showed high levels of activity of N-acetylglucosaminidase, alpha in the liver (600% of healthy levels) and a moderate increase (7% of healthy levels) in the levels of activity in serum. See
Example 16: Intracisternal Delivery of AAV9-CAG-hNaglu
[0169] A total dose of 9.310.sup.9 vector genomes of AAV9-CAG-hNaglu vector was injected into the cisterna magna of 2-month-old MPSIIIB animals in a total volume of 54
[0170] The intra-CSF administration of AAV9-CAG-hNaglu vectors led to high levels of N-acetylglucosaminidase, alpha activity in all brain areas analysed (50-100% of healthy mice); in the most forefront parts of the brain activity reached the levels observed in healthy animals. See
Example 17: Intracisternal Delivery of AAV9-CAG-cohNaglu
[0171] A total dose of 9.310.sup.9 vector genomes of AAV9-CAG-cohNaglu vector was injected into the cisterna magna of 2-month-old MPSIIIB animals in a total volume of 5 l.
[0172] Intracisternal administration of AAV9-CAG-cohNaglu vectors led a considerable increase in the levels of N-acetylglucosaminidase, alpha activity, which ranged from 22 to 45% of healthy values, in all brain regions analysed. See
Example 18: Hydrodynamic Delivery of the Plasmid pAAV9-CAG-cohNaglu-Version2
[0173] A total dose of 50 g of the plasmid pAAV9-CAG-cohNaglu-version2 carrying an expression cassette containing an optimized version (version2) of human N-acetylglucosaminidase, alpha were administered to 2-month-old MPSIIIB mice via tail hydrodynamic tail vein injection. As aforementioned, this technique targets expression of the delivered plasmid to the liver. See Liu et al., supra.
[0174] One week post plasmid delivery, N-acetylglucosaminidase, alpha activity was increased over pre-treatment levels in the liver and serum of all the animals that received a hydrodynamic injection of the plasmid containing the optimized version2 of N-acetylglucosaminidase, alpha-coding sequence. See
Example 19: Hydrodynamic Delivery of the Plasmid pAAV9-CAG-cohNaglu-Version3
[0175] A total dose of 50 g of the plasmid pAAV9-CAG-cohNaglu-version3 containing the a codon optimized version (version3) human N-acetylglucosaminidase, alpha expressing cassette were administered to 2-month-old MPSIIIB mice via tail hydrodynamic tail vein injection. As aforementioned, this technique targets expression of the delivered plasmid to the liver. See Liu et al., supra.
[0176] One week post plasmid delivery, a considerable increase in N-acetylglucosaminidase, alpha activity over pre-treatment levels was documented in the liver and serum of all the animals administered with the plasmid carrying the expression cassette that contained the version3 of codon-optimized human N-acetylglucosaminidase, alpha-coding sequence. See
Example 20: Intracisternal Delivery of AAV9-CAG-comNaglu
[0177] A total dose of 310.sup.10 vector genomes of AAV9-CAG-comNaglu vector was injected into the cisterna magna of 2-month-old MPSIIIB animals in a total volume of 10 l.
[0178] AAV9 vector genomes could be detected in all brain areas analysed, as well as in the spinal cord. In peripheral tissues, vector genomes could be detected at considerable gene copy numbers only in the liver, and at low gene copy numbers in the lymph nodes in which the head drains (mandibular lymph nodes). See
[0179] The intra-CSF administration of AAV9-CAG-comNaglu vectors led to very high levels of N-acetylglucosaminidase, alpha activity in all brain areas analysed; reaching levels that were several fold higher than those observed in healthy animals in all regions. See
[0180] In agreement with the correction of the lysosomal pathology, all signs of inflammation disappeared from the brains of treated MPSIIIB mice. The signal intensities for the stainings used to detect astrocytosis (GFAP) and microgliosis (BSI-B4) were similar in treated MPSIIIB mice and in healthy animals, as opposed to the signal documented in untreated MPSIIIB mice that showed a clear upregulation of these markers of neuroinflammation. See
[0181] AAV9 vectors administered to the CSF leak to the periphery and transduce the liver. See
[0182] The impact of the intra-CSF administration of AAV9-CAG-comNaglu on behaviour was assessed with the open field test, which evaluates the general locomotor and exploratory activity of mice in unknown surroundings. Untreated and AAV9-null-treated MPSIIIB mice displayed reduced exploratory activity compared with healthy mice in terms of the latency to enter the centre, the time spent in the border, the number of entries to the centre, the resting time, the total distance traveled, and the number of lines crossed. Intracisternal administration of AAV9-CAG-comNaglu completely corrected behavioural deficits. See
[0183] Furthermore, treatment with AAV9-CAG-comNaglu significantly extended the lifespan of MPSIIIB mice. By 15 months of age, all untreated MPSIIIB mice had died while 100% of the animals receiving intracisternal AAV9-CAG-comNaglu were still alive at 18 months of age. See
Example 21: Intracisternal Delivery of AAV9-CAG-cocNaglu to Dogs
[0184] The first step towards the clinical application of a gene therapy approach requires the demonstration of its feasibility in a large animal model. We previously demonstrated that the distribution of AAV9 vectors upon intra cerebrospinal fluid administration to Beagle dogs, an animal model with a brain size closer to that of humans, is very similar to that observed in mice receiving an equivalent dose of vector through the same route. See Haurigot et al., supra. Briefly, the administration of 210.sup.13 vg of AAV9 vectors encoding for a reporter protein GFP demonstrated widespread transduction of cells in the brain, cerebellum, meninges, spinal cord and dorsal root ganglia. Similar to the observations made in mice, GFP was also detected in the liver of Beagle dogs, where an average of 3.7% of hepatocytes was transduced. See Haurigot et al., supra. Importantly, the intra-CSF administration of AAV9 vectors encoding for the lysosomal enzyme sulfamidase, whose deficit causes MPSIIIA, led to sustained levels of enzyme in the CSF of treated dogs. The CSF bathes the CNS, making the enzyme available to different CNS structures. Indeed, the periodic delivery of recombinant enzyme to the CSF is a therapeutic strategy currently under clinical investigation for MPSIIIA. See NCT01155778 and NCT01299727, clinicaltrials.gov.
[0185] The same approach has been used to illustrate the potential efficacy of the AAV 9 vectors according to the present invention.
[0186] A total dose of 6.510.sup.12 vg AAV9-CAG-cocNaglu vectors was administered to the cisterna magna of 4 adult Beagle dogs (Dogs 1-4). To evaluate the impact of preexisting immunity on the CSF levels of N-acetylglucosaminidase, alpha activity that could be achieved by the treatment, two of those dogs (Dogs 3 and 4) were immunized by systemic administration of 110.sup.11 vg/kg of non-coding AAV9-null vectors 6 weeks before CSF delivery. By the time the intracisternal administration of vectors was performed, nave dogs had low titers of anti-AAV9 neutralizing antibodies (NAbs) in circulation and CSF, as it would be expected for animals that have not been previously exposed to the wild-type or recombinant virus. In contrast, pre-immunized dogs had high NAb titers in the circulation but low levels in the CSF, an observation compatible with the asymmetrical distribution of NAbs across the blood-brain barrier. See