ADENOASSOCIATED VIRUS VECTORS FOR THE TREATMENT OF MUCOPOLYSACCHARIDOSES
20180169272 · 2018-06-21
Inventors
- Maria Fàtima Bosch Tubert (Cerdanyola del Valles, ES)
- Virginia AREBA HAURIGOT (Barcelona, ES)
- Sandra MOTAS MALLOL (Foixá, ES)
Cpc classification
A61K9/0019
HUMAN NECESSITIES
C12N15/79
CHEMISTRY; METALLURGY
C12N2800/22
CHEMISTRY; METALLURGY
C12N2750/14143
CHEMISTRY; METALLURGY
A61K48/0075
HUMAN NECESSITIES
A61K48/0016
HUMAN NECESSITIES
A61K48/0066
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
International classification
A61K48/00
HUMAN NECESSITIES
C12N15/79
CHEMISTRY; METALLURGY
A61K9/00
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
Abstract
The present invention provides new Adeno-associated virus-derived vectors and pharmaceutical compositions containing the same for the treatment of lysosomal storage disorders and specially, for the treatment of mucopolysaccharidoses Type II.
Claims
1-22: (canceled)
23. An isolated nucleic acid encoding Iduronate-2-sulfatase (IDS) having the polypeptide sequence of SEQ ID NO:1, wherein the nucleic acid comprises a nucleotide sequence which exhibits between 75% and 90% identity with SEQ ID NO:2.
24. The isolated nucleic acid of claim 23, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:5.
25. The isolated nucleic acid of claim 23, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:8.
26. A plasmid comprising a nucleic acid encoding IDS, wherein the nucleic acid comprises a nucleotide sequence which exhibits at least 75% identity with the sequence of SEQ ID NO:2.
27. The plasmid of claim 26, wherein the nucleic acid comprises a nucleotide sequence which exhibits between 75% and 90% identity with SEQ ID NO:2.
28. The plasmid of claim 26, wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8.
29. The plasmid of claim 26 which is pAAV-CAG-hIDS deposited under accession number DSM 29866, and having a nucleotide sequence as set forth in SEQ ID NO:3.
30. The plasmid of claim 26 which is pAAV-CAG-ohIDS-version1 deposited under accession number DSM 29867, and having a nucleotide sequence as set forth in SEQ ID NO:6.
31. The plasmid of claim 26, which is pAAV-CAG-ohIDS-version2 deposited under accession number DSM 29868, and having a nucleotide sequence as set forth in SEQ ID NO:9.
32. A recombinant vector comprising a nucleic acid encoding IDS, wherein the nucleic acid comprises a nucleotide sequence which exhibits at least 75% identity with SEQ ID NO:2.
33. The recombinant vector of claim 32, wherein the nucleic acid comprises a nucleotide sequence which exhibits between 75% and 90% identity with SEQ ID NO:2.
34. The recombinant vector of claim 32, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:5.
35. The recombinant vector of claim 32, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:8.
36. The recombinant vector of claim 32, wherein the vector is an Adeno-associated Virus Vector of serotype 9 (AAV9).
37. The recombinant vector of claim 32, wherein the nucleotide sequence is linked to a CAG promoter as set forth in SEQ ID NO:14.
38. The recombinant vector of claim 32, which is AAV9-CAG-hIDS comprising the nucleotide sequence of SEQ ID NO:2 which is linked to the CAG promoter of SEQ ID NO:14.
39. The recombinant vector of claim 32, which is AAV9-CAG-ohIDS-version1 comprising the nucleotide sequence SEQ ID NO:5 which is linked to the CAG promoter of SEQ ID NO:14.
40. The recombinant vector of claim 32, which is AAV9-CAG-ohIDS-version2 comprising the nucleotide sequence SEQ ID NO:8 which is linked to the CAG promoter of SEQ ID NO:14.
41. A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid encoding IDS of claim 23.
42. A pharmaceutical composition comprising a therapeutically effective amount of the plasmid of claim 26.
43. A pharmaceutical composition comprising a therapeutically effective amount of the recombinant vector of claim 32.
44. The pharmaceutical composition of claim 43 which is in a form for intravenous or intracisternal administration.
45. A method for treating mucopolysaccharidosis type II in a subject in need thereof, the method comprising administering an effective amount of the nucleic acid of claim 23.
46. A method for increasing Iduronate-2-sulfatase (IDS) activity in a subject in need thereof, the method comprising administering an effective amount of the nucleic acid of claim 23.
47. A method of producing the recombinant vector of claim 32, comprising the steps of: i) providing a first vector comprising a sequence coding for a protein of interest interposed between a first Adeno-associated virus (AAV) terminal repeat and a second AAV terminal repeat, a CAG promoter operably linked to the sequence coding for a 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; 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).
48. A method for the preparation of the plasmid of claim 26, comprising the step of: i) excising the sequence coding for a protein of interest from the starting plasmid, by digestion, in particular using MluI/EcoRI, 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.
49. An isolated cell comprising a nucleic acid encoding iduronate-2-sulfatase (IDS) having the polypeptide sequence of SEQ ID NO:1, wherein the nucleic acid comprises a nucleotide sequence which exhibits at least 75% identity with SEQ ID NO:2.
50. The isolated cell of claim 49, wherein the nucleic acid comprises a nucleotide sequence exhibits between 75% and 90% identity with SEQ ID NO:2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DEPOSIT OF MICROORGANISMS
[0051] The plasmids pAAV-CAG-hIDS (SEQ ID NO: 3), pAAV-CAG-ohIDS-version1 (SEQ ID NO: 6) and pAAV-CAG-ohIDS-version2 (SEQ ID NO: 9) were deposited on Dec. 18, 2014, under access number DSM 29866, DSM 29867 and DSM 29868 at the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffenstrae 7 B, D-38124 Braunschweig, Federal Republic of Germany.
Definitions
[0052] The term nucleotide sequence or isolated 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.
[0053] The term % sequence identity or % identity refer 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.
[0054] Herein, the % sequence identity or % 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.
[0055] 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.
[0056] 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. Glycosylation of a protein is the addition of different carbohydrates that are linked covalently to the amino acid chain.
[0057] 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 iduronate-2-sulfatase (IDS) 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] The term gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a cell 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.
[0062] The term recombinant viral vector or viral vector refers 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.
[0063] 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
[0064] The present invention provides new nucleotide sequences for the treatment of mucopolysaccharidoses, in particular mucopolysaccharidoses type II (MPSII), or Hunter syndrome.
[0065] The nucleotide sequences according to the present invention codify for the protein Iduronate-2-sulfatase (referred to as IDS) as set forth in SEQ ID NO:1, enzyme involved in the stepwise degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. The natural IDS or wild type IDS terms refer in the context of the invention to a nucleotide sequence obtained or produced from host cells using methods known to those skilled in the art, or alternatively chemically synthetized using as starting material the coding sequence (CDS) for IDS of each species. Preferably, natural IDS or wild type IDS are chemically synthetized from the murine CDS (referred to as mIDS) or from the humans CDS (referred to as hIDS). More preferably the nucleotide sequence hIDS is chemically synthetized from human CDS and corresponds to SEQ ID NO: 2.
[0066] According to the present invention, evidence establishes that modified nucleotide sequences, also referred to as optimized nucleotide sequences, mediate the production of greater amounts of protein of interest, likely due to efficient transcription of mRNA or the transcription of a more stable mRNA, or the more efficient usage of codons. These sequences are referred herein also as optimized sequences; omIDS and ohIDS, when obtained from murine and human CDS, respectively. Advantageously, optimized sequences according to the present invention are codon optimized sequences.
[0067] Thus a first aspect of the invention relates to an isolated nucleotide sequence coding for the protein Iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1 and having between 75% and 90% identity with SEQ ID NO:2. In particular, the isolated nucleotide sequence according to the invention has 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87% or 90% identity with SEQ ID NO:2. In a preferred embodiment, the isolated nucleotide sequences of the present invention are selected from ohIDS-version1 as set forth in SEQ ID NO:5 and ohIDS-version2 as set forth in SEQ ID NO:8.
[0068] In accordance with the present invention, the isolated sequences described hereinbefore may be inserted into a multiple cloning site (MCS) of a backbone plasmid. In particular the backbone plasmid is a plasmid that contains the ITRs of the Adeno-associated Virus (AAV), referred to as pAAV herein.
[0069] A second aspect of the invention provides a plasmid containing a nucleotide sequence coding for the protein Iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1 and having at least 75% identity with SEQ ID NO:2. Advantageously, the plasmid according to the present invention has between 75% and 90% identity with SEQ ID NO:2. In particular, the plasmid according to the invention contains an isolated nucleotide sequence having 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87% or 90% identity with SEQ ID NO:2. In a preferred embodiment, the plasmid of the present invention contains a nucleotide sequence selected from hIDS as set forth in SEQ ID NO:2, ohIDS-version1 as set forth in SEQ ID NO:5 and ohIDS-version2 as set forth in SEQ ID NO:8.
[0070] In addition to the isolated sequences mentioned above, the plasmids according to the present invention also include conventional control elements which may be operably linked to the nucleotide sequence in a way that allows its transcription, translation and/or expression in a cell transfected with the plasmids. In particular, the plasmids according to the present invention contain a promoter as control element operably linked to the nucleotide sequence of interest. A great number of promoters, which are native or chimeric, constitutive or inducible, ubiquitous and/or tissue-specific are known in the art. Advantageously, the promoter used in the context of the present invention is the CAG promoter which refers to the combination comprising the cytomegalovirus early enhancer element and the chicken B-actin promoter. It further includes portions of the chicken B-actin and rabbit B-globin introns that confer stability to the mRNA derived from the nucleotide sequence of interest, See Alexopoulou A, et al., BMC Cell Biology 2008; 9(2): 1-11. The CAG promoter included in the pAAV plasmids of the present invention has a sequence SEQ ID NO:14. This specific CAG promoter allows a long-lasting expression of the missing enzyme in all areas of the brain and in the liver. As a consequence the lysosomal accumulation of glycosaminoglycan (GAG) is corrected, preventing in this way the neurological and somatic alterations characteristic of MPSII.
[0071] In a particularly advantageous embodiment, the plasmid according to the invention is the plasmid pAAV-CAG-hIDS, as set forth in SEQ ID NO:3 with access number DSM 29866.
[0072] In another particularly advantageous embodiment, the plasmid according to the invention is the plasmid pAAV-CAG-ohIDS-version1, as set forth in SEQ ID NO:6 with access number DSM 29867.
[0073] In another particularly advantageous embodiment, the plasmid according to the invention is the plasmid pAAV-CAG-ohIDS-version2, as set forth in SEQ ID NO:9 with access number DSM 29868.
[0074] A third aspect of the invention relates to new recombinant vectors for the treatment of mucopolysaccharidoses type II. It has to be understood that a vector of the present invention is a capsid protein as well as a vector genome contained within, used to transfer a genetic material of interest into a cell. Apart from said genetic material of interest, the genome of the vector may also contain different functional elements that include control elements for transcription such as promoters or operators, transcription factors binding regions or enhancers and control elements for the initiation or termination of translation.
[0075] The vectors according to the invention are derived from Adeno-associated viruses (AAV) and are used to transfer the nucleotide sequence of interest into a target cell. They have proved to have a high efficiency in transducing post-mitotic cells in a wide range of tissues. In the context of the present invention, the vectors are used to deliver the human Iduronate-2-sulfatase coding sequence (hIDS of SEQ ID NO: 2) or an optimized version of the human Iduronate-2-sulfatase coding sequence, (ohIDS-version1 of SEQ ID NO: 5 or ohIDS-version2 of SEQ ID NO: 8). An adeno-associated vector is a vector derived from an adeno-associated virus of the family of parvoviridae. The adenoassociated virus genome is built of single-stranded deoxyribonucleic acid (ssDNA). These viruses 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 adeno-associated 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 AAV has a serotype 1, 2, 5, 7, 8, 9 or 10. Preferably, the AAV is of serotype 9 (AAV9), since it shows the best ability to deliver the genetic material to the brain as well as to peripheral organs upon a single administration to the CSF. The 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 of the human Iduronate-2-sulfatase (hIDS) gene or an optimized version of it (also referred to as nucleotide sequence according to the present invention), and the polyA from the rabbit beta-globin gene.
[0076] Thus in this aspect the invention relates to a recombinant AAV containing a nucleotide sequence coding for the protein Iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1 and having at least 75% identity with SEQ ID NO:2. Advantageously, the recombinant AAV according to the invention contains between 75% and 90% identity with SEQ ID NO:2. In particular the recombinant AAV according to the invention contains a nucleotide sequence according to the invention having 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87% or 90% identity with SEQ ID NO:2. In a preferred embodiment, the isolated nucleotide sequences contained in the recombinant AAV of the present invention are selected from hIDS as set forth in SEQ ID NO:2, ohIDS-version1 as set forth in SEQ ID NO:5 and ohIDS-version2 as set forth in SEQ ID NO:8.
[0077] In an advantageous embodiment of this aspect, the present invention relates to a recombinant AAV9 containing a nucleotide sequence coding for the protein Iduronate-2-sulfatase IDS as set forth in SEQ ID NO:1 and having at least 75% identity with SEQ ID NO:2. Preferably, the recombinant AAV9 of the invention contains a nucleotide sequence having between 75% and 90% identity with SEQ ID NO:2. In particular the recombinant AAV9 of the invention contain a nucleotide sequence according to the invention having 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87% or 90% identity with SEQ ID NO:2. In a preferred embodiment, the recombinant AAV9 of the invention contains the nucleotide sequences selected from hIDS as set forth in SEQ ID NO:2, ohIDS-version1 as set forth in SEQ ID NO:5 and ohIDS-version2 as set forth in SEQ ID NO:8.
[0078] It has further been surprisingly found that the association, in the same entity, of the AAV9 capsid with a nucleotide sequence coding for the Iduronate-2-sulfatase (IDS), together with a chosen promoter, especially the CAG promoter, allows a long-lasting expression of the missing enzyme in all areas of the brain, in particular when the entity is delivered to the cerebrospinal fluid (CSF) through intracisternal injection. As a consequence the lysosomal accumulation of glycosaminoglycan (GAG) is corrected, preventing by that way the neurological alterations characteristic of the MSPII disease. This effect has been observed even in the olfactory bulb, which is distant from the point of administration of the vectors (cistema magna). Further the AAV9 vectors according to the invention delivered into the CSF 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 Iduronate-2-sulfatase (IDS) 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.
[0079] Accordingly the present invention relates to AAV9 vectors containing a CAG promoter linked to a nucleotide sequence coding for protein Iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1.
[0080] In particular the AAV9 vectors of the present invention contain a CAG promoter linked to a nucleotide sequence coding for the protein iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1 and having at least 75% identity with SEQ ID NO:2. Advantageously, the AAV9 vectors of the invention contain a CAG promoter linked to a nucleotide sequence coding for the protein iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1 and having between 75% and 90% identity with SEQ ID NO:2. In particular the nucleotide sequence contained in the AAV9 vector according to the invention has 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87% or 90% identity with SEQ ID NO:2.
[0081] In a preferred embodiment, the recombinant vector of the present invention is the AAV9-CAG-hIDS (SEQ ID NO: 4) containing the nucleotide sequence SEQ ID NO:2 operably linked to the CAG promoter of SEQ ID NO:14.
[0082] In another preferred embodiment, the recombinant vector of the present invention is the AAV9-CAG-ohIDS-version1 (SEQ ID NO: 7) containing the nucleotide sequence SEQ ID NO:5 operably linked to the CAG promoter of SEQ ID NO:14.
[0083] In another preferred embodiment, the recombinant vector of the present invention is the AAV9-CAG-ohIDS-version2 (SEQ ID NO: 10) containing the nucleotide sequence SEQ ID NO:8 operably linked to the CAG promoter of SEQ ID NO:14.
[0084] The recombinant vectors of the invention as defined hereinbefore may be obtained from the corresponding plasmids also described hereinbefore by transfection of HEK293 cells using methods known in the state of the art.
[0085] Thus the present invention further provides a method for the production of the adenoassociated viral vectors AAV according to the invention, and especially a AAV9. The process comprises the steps of: [0086] i) providing a first plasmid 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, and a third vector comprising the adenovirus helper function genes; [0087] ii) co-transfection of competent cells with the vectors of step i); [0088] iii) culture of the transfected cells of step ii) for a period of time sufficient to produce viral particles; and [0089] iv) purification of the vectors from the culture of step iii).
[0090] 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. In another preferred embodiment, the AAV cap genes of the second vector are from the AAV serotype 9.
[0091] The invention also provides a method for the preparation of the plasmid according to the invention, comprising the steps of: [0092] i) excising the sequence coding for the protein of interest from the starting plasmid, by digestion, in particular using MluI/EcoRI, [0093] 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.
[0094] The present invention contemplates, in an additional aspect, pharmaceutical compositions containing a therapeutically effective amount of the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors, described herein.
[0095] Pharmaceutical compositions of the invention comprise the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors described herein 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; hydrophilic polymers; amino acids; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents; sugar alcohols such as mannitol or sorbitol, salt forming counterions such as sodium; and/or non-ionic surfactants such as polyethylene-polyoxypropylene block copolymer (Pluronic F68) and polyethylene glycol (PEG).
[0096] 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.
[0097] The 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.
[0098] Still a further aspect of the present invention relates to the therapeutical use of the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors, described herein. As mentioned above, the isolated nucleotide sequences, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors according to the invention mediate expression of the missing IDS enzyme, thus correcting the lysosomal accumulation of GAGs. This allows correcting all clinical signs of mucopolysaccharidoses type II (MPSII). In this respect, the present invention also concerns the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors, described herein for use as a medicament.
[0099] In particular, the invention relates to the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors, described herein for increasing iduronate-2-sulfatase (IDS) activity in the body.
[0100] In a further preferred aspect, the present invention relates to the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors, described herein for treatment of mucopolysaccharidoses type II (MPSII).
[0101] In a still further embodiment, the present invention relates to the use of the isolated nucleotide sequences described herein, the plasmids as described herein, or the AAV vectors, especially AAV9 vectors, described herein for the manufacture of a medicament useful for the treatment of mucopolysaccharidoses type II (MPSII).
[0102] Another embodiment of the present invention is directed to the method of treatment of mucopolysaccharidoses type II (MPSII), comprising the step of administering at least an isolated nucleotide sequences described herein, a plasmid as described herein, or an AAV vectors, especially AAV9 vectors, described herein to a subject in need thereof.
[0103] The present invention further provides an isolated cell comprising the nucleotide sequence coding for Iduronate-2-sulfatase SEQ ID NO: 1. In particular the cell according to the invention comprises a nucleotide sequence coding for the protein Iduronate-2-sulfatase (IDS) as set forth in SEQ ID NO:1 and having at least 75% identity with SEQ ID NO:2, and advantageously between 75% and 90% identity with SEQ ID NO:2. In particular the nucleotide sequence contained in the isolated cell according to the invention has 75%, 76%, 77%, 78%, 79%, 80%, 82%, 85%, 87% or 90% identity with SEQ ID NO:2.
[0104] In a preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 2 coding for Iduronate-2-sulfatase (IDS) SEQ ID NO: 1.
[0105] In another preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 5 coding for Iduronate-2-sulfatase (IDS) SEQ ID NO: 1.
[0106] In another preferred embodiment the cells of the invention comprise the nucleotide sequence SEQ ID NO: 8 coding for Iduronate-2-sulfatase (IDS) SEQ ID NO: 1.
[0107] The following examples are merely illustrative of certain embodiments of the invention and cannot be considered as restricting in any way.
General Procedures
[0108] 1. Recombinant AAV Vectors
[0109] The AAV vectors described herein were obtained by triple transfection. The materials required for making the vectors were: HEK293 cells (expressing adenoviral E1 genes), helper plasmid providing adenovirus function, plasmid providing AAV rep genes from serotype 2 and cap genes from serotype 9 (AAV9) and, finally, the backbone plasmid with AAV2 ITRs and the construct of interest.
[0110] To generate Iduronate-2-sulfatase-expressing AAV vectors, the optimized or non-optimized coding sequences of human or murine Iduronate-2-sulfatase were cloned into an AAV backbone plasmid under the control of the ubiquitous hybrid CAG promoter. Large-scale production of plasmids was done using an EndoFree Plasmid Megaprep Kit (Qiagen).
[0111] 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+0.001% Pluronic F68, filtered, titred by qPCR and stored at 80 C. until use.
[0112] The vectors of the present invention were constructed according to molecular biology techniques well known in the art.
[0113] 2. Animals
[0114] An Iduronate-2-sulfatase-deficient mouse (MPSII) model was purchased from Taconic (Germantown, N.Y. 12526 USA, Stock TF1838). Affected MPSII and healthy control mice were inbred from hemizygous males and heterozygous female 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-TTT TGT GTA CTC CAA CCC CG-3 (SEQ ID NO:15), Reverse Primer: 5-TGT CTC CAT AAC AGC CCA GG-3 (SEQ ID NO:16), Reverse Primer Mutation: 5-GCC CTC ACA TTG CCA AAG GA-3 (SEQ ID NO:17). 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.).
[0115] 3. Hydrodynamic Delivery of IDS-Encoding Plasmids to Mice
[0116] For hydrodynamic delivery of pAAV-CAG-hIDS, pAAV-CAG-ohIDS-version1 and pAAV-CAG-ohIDS-version2 plasmids, 3-month-old MPSII and wild-type animals received through tail vein injection in <5 seconds a total dose of 30 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 divided into two cohorts, and sacrificed either at 48 hours or 1 week after hydrodynamic injection of the plasmids. Organs were harvested as described in the following section.
[0117] 4. Vector Administration to Mice
[0118] For intra-CSF delivery of AAV9-CAG-omIDS vectors to mice, a total dose of 510.sup.10 vg were injected to the cistema magna of 2-month-old MPSII animals. A similar cohort of animals was injected with 510.sup.10 vg control non-coding (AAV9-Null) vector. At 6 and 10 months of age, i.e. 4 and 8 months post vector administration, mice were anesthetized and tissues were harvested.
[0119] For the intravenous delivery of AAV9 vectors containing the wild-type hIDS or either of the optimized versions of the IDS coding sequence to mice, a total dose of 110.sup.10 vg of vector were injected in the tail vein of 3.5-month-old MPSII animals. WT and untreated MPSII animals served as controls. Three weeks after vector administration, mice were anesthetized and tissues were harvested.
[0120] 5. Sample Collection
[0121] At sacrifice, animals were deeply anesthetized and then transcardially perfused with 12 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, testicles, urinary bladder, intestine and adipose tissue) were collected and either frozen in liquid nitrogen and stored at 80 C. or immersed in formalin for subsequent histological analyses.
[0122] 6. Iduronate-2-Sulfatase Activity and Glycosaminoglycan Quantification
[0123] Brain, liver, lung and heart samples were sonicated in Mili-Q water. Serum was analysed unprocessed. Iduronate-2-sulfatase activity was determined with a 4-methylumbelliferone-derived fluorogenic substrate (Moscerdam Substrates, Oegstgeest, NL), as described previously. See Voznyi et al., J Inher Metab Diss 2001; 24:675-680. Brain, liver, lung and heart 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.
[0124] 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.
[0125] 7. Activity of Other Lysosomal Enzymes
[0126] Brain and liver samples were sonicated in 500 l of Mili-Q water and enzyme activities were determined in supernatants using 4-methylumbelliferone-derived fluorogenic substrates. Serum was analysed unprocessed. IDUA activity was assayed in 15 g of protein incubated for 1 h at 37 C. with 4-methylumbelliferyl -L-iduronide (Glycosynth). See Bacter et al., Blood 2002; 99(5)1857-9. SGSH activity was measured as previously described. See Karpova et al., J Inherit Metab Dis. 1996; 19(3):278-285, Haurigot et al., supra. Briefly, 30 g of protein were first incubated with 4-MU-GlcNS for 17 hours at 47 C. The second incubation was carried out in the presence of 10 U/ml of -glucosidase (Sigma-Aldrich) in 0.2% BSA for 24 hours at 37 C. For NAGLU activity, 30 g of tissue protein extract were incubated with 4-methylumbelliferyl--N-acetyl-D-glucosaminide (Moscerdam Substrates) for 3 h at 37 C., as previously described. See Marsh et al., Clin Genet. 1985; 27(3):258-62, Ribera et al., supra. HGSNAT activity was determined from 30 g of protein extract incubated with Acetylcoenzyme A and 4-methylumbelliferyl--D-glucosamine (MU-GlcNH.sub.2, Moscerdam Substrates) for 17 h at 37 C. See Voznyi et al., J Inh Metab Dis 1993; 16:465-72. GALNS activity was assayed by a 2-step protocol using 10 g of protein extract and 4-Methylumbelliferyl -D-Galactopyranoside-6-sulfate Sodium Sal (MU-Gal-6S) during the first incubation for 17 h at 37 C. The second step was carried out adding P.sub.i-buffer (0.9M Na.sub.2HPO.sub.4/0.9M NaH.sub.2PO.sub.4 buffer, pH4.3+0.02% (w/v) Na-azide) and -Galactosidase (-Gal-Ao, Sigma) and incubating the mix for 2 h at 37 C. See van Diggelen et al., Clin Chim Acta 1990; 187:131-40. The activity of GUSB enzyme was determined from 10 g of protein extract 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 extract with 4-methylumbelliferyl N-acetyl--D-glucosaminide (Sigma) for 1 h at 37 C. After stopping reactions by increasing the pH, released fluorescence was measured with FLx800 fluorimeter (BioTek Instruments). All brain and liver activities levels were normalized against the total amount of protein, quantified using Bradford protein assay (Bio-Rad, Hercules, Calif., US).
[0127] 8. Histological Analysis
[0128] Tissues were fixed for 12-24 h in formalin, embedded in paraffin and sectioned. For immunohistochemical detection of LAMP2 in brain, paraffin sections were subjected to heat-induced epitope retrieval in citrate buffer, pH 6, and then incubated overnight at 4 C. with rat anti-LAMP2 antibody (Ab13524; Abcam, Cambridge, UK) diluted at 1:500 and subsequently incubated with biotinylated rabbit anti-rat antibody (Dako, Glostrup, DK) 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. LAMP2, 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.
[0129] 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).
[0130] The NIS Elements Advanced Research 2.20 software was used to quantify LAMP2, GFAP, and BSI-B4 signals in 3-4 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.
[0131] 9. Quantification of Vector Genome Copy Number in Tissues
[0132] After an overnight tissue digestion in Proteinase K (0.2 mg/ml), total DNA was isolated with MasterPureDNA Purification Kit (Epicenter). Quantitative PCR with primers and probe specific for the HBB2 sequence (contained in the polyA segment) were used to quantify the vector genome copy numbers in 20 ng of total DNA. Forward primer: 5-CTT GAG CAT CTG ACT TCT GGC TAA T-3; reverse primer: 5-GAT TTG CCC TCC CAT ATG TCC-3; probe: 5-CCG AGT GAG AGA CAC AAA AAA TTC CAA CAC-3. Reference standard curve built by serial dilutions of the linearized plasmid containing polyA sequence was used to interpolate the final values of vg/sample.
[0133] 10. Open Field Test
[0134] The behavior of 6-month-old mice was analyzed by the Open Field test performed between 9:00 am and 1:00 pm. Animals were placed in the lower left corner of a brightly lit chamber (414130 cm) crossed by 2 bundles of photobeams (SedaCom32; Panlab) that detected horizontal and vertical movements of the mice. The area surface was divided into three squared concentric regions: center (1414 cm), periphery (2727 cm) and border (4141 cm). Exploratory and motor activities were recorded during the first 3 minutes of the test using a video-tracking system (SmartJunior, Panlab).
[0135] 11. Statistical Analysis
[0136] All results are expressed as meanSEM. Statistical comparisons were made using one-way ANOVA. Multiple comparisons between control and treatment groups will be made using Dunnett's post test, and between all groups using Tukey's post test. Statistical significance was considered if P<0.05. Kaplan-Meier curves were used to estimate survival and the long-rank test was used for comparisons.
EXAMPLES
Example 1: Construction of pAAV-CAG-hIDS
[0137] The CDS for human Iduronate-2-sulfatase was utilized as starting material (NCBI Reference Sequence: NM_000202.6) and chemically synthetized for this purpose (GenScript Inc). The CDS was received cloned inside the plasmid pUC57_(AmpR) flanked by Swal restriction sites.
[0138] The Swal-Swal human Iduronate-2-sulfatase CDS fragment was excised from the pUC57 plasmid and subsequently cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG after rendering the 5 and 3 overhangs blunt with Klenow fragment (Fermentas). The resulting plasmid was named pAAV-CAG-hIDS (accession number DSM 29866). See
[0139] 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: Construction of pAAV-CAG-ohIDS-Version1
[0140] Expression cassettes including an optimized version of human Iduronate-2-sulfatase cDNA sequence (ohIDS) were designed and obtained. The sequence optimization was performed to maximize the efficiency of Iduronate-2-sulfatase 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 CDS for human Iduronate-2-sulfatase (NCBI Reference Sequence: NM_000202.6) was used as starting point for sequence optimization (DNA 2.0 Inc). The optimized CDS was received cloned inside the plasmid pJ204:191476 (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0141] The MluI/EcoRI optimized human Iduronate-2-sulfatase CDS fragment was excised from the pJ204:191476 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-ohIDS-version1 (accession number DSM 29867). See
Example 3: Construction of pAAV-CAG-ohIDS-Version2
[0142] The CDS for human Iduronate-2-sulfatase (NCBI Reference Sequence: NM_000202.6) was subjected to sequence optimization (GeneScript Inc). The optimized CDS was received cloned inside the plasmid pUC57 (AmpR) flanked by MluI and EcoRI restriction sites at 5 and 3, respectively.
[0143] The pUC57-ohIDS plasmid was digested with MluI and EcoRI to excise the optimized Iduronate-2-sulfatase CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-ohIDS-version2 plasmid (accession number DSM 29868). See
Example 4: Construction of pAAV-CAG-omIDS
[0144] The CDS for murine Iduronate-2-sulfatase (NCBI Reference Sequence: NM_010498.3) 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.
[0145] The MluI/EcoRI optimized murine Iduronate-2-sulfatase CDS fragment (SEQ ID NO:11) 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-omIDS. See
Example 5: Production of AAV9-CAG-hIDS
[0146] Vectors AAV9-CAG-hIDS (SEQ ID NO:4) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita et al., Gene Ther. 1998; 5(7):938-45, Wright et al., Mol Ther. 2005; 12(1)171-8. 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 AAV2 ITRs (pAAV-CAG-hIDS); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); 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 et al., Gene Ther. 2010; 17(4):503-10. Vectors were dialyzed against PBS+0.001% Pluronic F68, filtered, titred by qPCR and stored at 80 C. until use. See
Example 6: Production of AAV9-CAG-ohIDS-Version1
[0147] Vectors AAV9-CAG-ohIDS-version1 (SEQ ID NO:7) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita et al., and Wright et al., supra. 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 AAV2 ITRs (pAAV-CAG-ohIDS-version1); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); 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 et al., supra. Vectors were dialyzed against PBS+0.001% Pluronic F68, filtered, titred by qPCR and stored at 80 C. until use. See
Example 7: Production of AAV9-CAG-ohIDS-Version2
[0148] Vectors AAV9-CAG-ohIDS-version2 (SEQ ID NO:10) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita et al., and Wright et al., supra. 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 AAV2 ITRs (pAAV-CAG-ohIDS-version2); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); 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 et al., supra. Vectors were dialyzed against PBS+0.001% Pluronic F68, filtered, titred by qPCR and stored at 80 C. until use. See
Example 8: Production of AAV9-CAG-omIDS
[0149] Vectors AAV9-CAG-omIDS (SEQ ID NO:13) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita et al., and Wright et al., supra. 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 AAV2 ITRs (pAAV-CAG-omIDS); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); 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 et al., supra. Vectors were dialyzed against PBS+0.001% Pluronic F68, filtered, titred by qPCR and stored at 80 C. until use. See
Example 9: Hydrodynamic Injection of pAAV-CAG-hIDS, pAAV-CAG-ohIDS-Version1 and pAAV-CAG-ohIDS-Version2 to Healthy Mice
[0150] A total dose of 30 lag of the plasmids pAAV-CAG-hIDS, pAAV-CAG-ohIDS-version1 and pAAV-CAG-ohIDS-version2 containing different versions of the Iduronate-2-sulfatase expressing cassette were administered to 2-month-old WT mice via tail hydrodynamic tail vein injection. This technique targets expression of the delivered plasmid to the liver. See Liu et al., Gene Ther. 1990; 6(7):1258-66.
[0151] Forty-eight hours post plasmid delivery, a considerable increase over basal levels in Iduronate-2-sulfatase activity was documented in the livers and serums of all the animals administered with Iduronate-2-sulfatase-coding plasmids. In both liver and serum, the levels of activity reached with the expression cassettes containing optimized versions of the Iduronate-2-sulfatase gene were higher than those obtained with the wild-type gene. Furthermore, in serum, the animals that received the pAAV-CAG-ohIDS-version2 plasmid showed levels of Iduronate-2-sulfatase activity that were statistically higher than those documented with the other 2 plasmids. See
Example 10: Hydrodynamic Injection of pAAV-CAG-hIDS, pAAV-CAG-ohIDS-Version1 and pAAV-CAG-ohIDS-Version2 to MPSII Mice
[0152] A total dose of 30 g of the plasmids pAAV-CAG-hIDS, pAAV-CAG-ohIDS-version1 and pAAV-CAG-ohIDS-version2 containing different versions of the Iduronate-2-sulfatase expressing cassette were administered to 3-month-old MPSII-affected mice via tail hydrodynamic tail vein injection.
[0153] Tissues were harvested 1 week after plasmid delivery. All three Iduronate-2-sulfatase-containing plasmids mediated a substantial increase in Iduronate-2-sulfatase activity with respect to MPSII animals that received saline injection; activities ranged from 1200% to 2200% of WT levels in liver and 2000% to 5700% of WT in serum. The levels of activity reached with the expression cassettes containing codon-optimized versions of the Iduronate-2-sulfatase gene were statistically higher than those mediated by the plasmid containing the wild-type gene. See
[0154] Consistent with the high levels of Iduronate-2-sulfatase activity documented in liver and serum, GAG content was completely normalized in all tissues analysed with all plasmid constructs. See
Example 11: Intravenous Delivery of AAV9-CAG-hIDS, AAV9-CAG-ohIDS-Version1 and AAV9-CAG-ohIDS-Version2 to MPSII Mice
[0155] MPSII mice (3.5-month-old) received an intravenous injection through the tail vein of 110.sup.10 vg of AAV9 vectors containing either the wild-type or the optimized human Iduronate-2-sulfatase sequences. Separate cohorts of age-matched WT and untreated MPSII mice served as controls. Three weeks after the treatment, animals were sacrificed and blood and liver samples were collected and analysed.
[0156] The intravenous administration of AAV9 vectors at a dose of 110.sup.10 vg/mouse targets transgene expression mainly to the liver. See Wu et al., Mol. Ther. 2006; 14(3):316-27, Inagaki et al., Mol. Ther. 2006; 14(1):45-33. When the activity of Iduronate-2-sulfatase was measured in liver extracts 3 weeks after vector delivery, a clear increase in enzymatic activity was documented in MPSII males that received any of the human IDS-coding vectors. See
[0157] Iduronate-2-sulfatase is a secretable lysosomal enzyme, and as such its expression in the liver is a source of circulating enzyme. See Haurigot et al, supra. Similar to the observations made in liver extracts, the levels of Iduronate-2-sulfatase activity achieved in circulation of MPSII animals treated with the AAV9 vectors containing the optimized human IDS sequences were several fold higher than those observed in healthy animals or in MPSII animals treated with vectors containing the wild-type human IDS sequence. See
[0158] Consistent with the high levels of Iduronate-2-sulfatase activity documented in liver and serum, GAG content was completely normalized in the liver of the MPSII mice treated with AAV9-CAG-ohIDS-version1 and AAV9-CAG-ohIDS-version2. GAG levels were, however, only partially reduced in the liver of animals treated with an equal dose of AAV9 vectors carrying the wild-type IDS coding sequence. See
Example 12: Intracisternal Delivery of AAV9-CAG-hIDS, AAV9-CAG-ohIDS-Version1 and AAV9-CAG-ohIDS-Version 2 to MPSII Mice
[0159] Two-month-old MPSII mice received an intracisternal injection of 510.sup.10 vg of AAV9 vectors containing either the wild-type or the optimized human Iduronate-2-sulfatase sequence in a total volume of 5 p1. Separate cohorts of age-matched WT, untreated MPSII mice and MPSII mice receiving 510.sup.10 vg of a non-coding vector (AAV9-CAG-Null) served as controls. At 3.5 months of age, i.e. 1.5 months after treatment, animals were sacrificed and samples were collected and analysed.
[0160] When the activity of Iduronate-2-sulfatase was measured in brain extracts, a clear increase in enzymatic activity was documented in MPSII males that received any of the human IDS-coding vectors. See
[0161] In agreement with the increase in IDS activity throughout the brain, the accumulation of substrate that characterizes the disease was corrected in the brains of treated MPSII mice, as indicated by the significant reduction in the GAG content. See
[0162] AAV9 vectors administered to the CSF leak to the periphery and transduce the liver. See Haurigot et al., Clin Invest. 2013; 123(8):3254-3271, Ribera et al., Hum Mol Genet. 2014; 24(7):2078-2095. Accordingly, an increase in Iduronate-2-sulfatase activity was documented in the liver and serum of MPSII mice treated with any of the human IDS-coding vectors. See
Example 13: Intracisternal Delivery of AAV9-CAG-omIDS
[0163] A total dose of 510.sup.10 vector genomes of AAV9-CAG-omIDS vector was injected into the cistema magna of 2-month-old MPSII animals in a total volume of 5 p1. First, mice were analysed at 6 months of age, i.e. 4 months after vector administration. The intra-CSF delivery of AAV9-CAG-omIDS vectors led to restoration of Iduronate-2-sulfatase activity in all brain areas analysed, reaching levels that averaged 40% of those observed in healthy animals in the different regions. See
[0164] The disruption of normal lysosomal homeostasis due to the accumulation of undegraded substrate can alter the activity of other lysosomal enzymes different from the one directly affected by the mutation. See Ribera et al., Hum Mol Genet. 2014; doi: 10.1093/hmg/ddu727. In the brains of untreated MPSII mice or MPSII mice treated with control Null vector, the activities of IDUA (iduronidase, alpha-L-), SGSH (N-sulfoglucosamine sulfohydrolase), NAGLU (N-acetylglucosaminidase, alpha), HGSNAT (heparan-alpha-glucosaminide N-acetyltransferase), GALNS (galactosamine (N-acetyl)-6-sulfatase), GUSB (glucuronidase, beta), HEXB (hexosaminidase B) were altered, but treatment with AAV9-CAG-omIDS returned those activities to the levels observed in healthy WT animals indicating that the vector was capable of restoring lysosomal homeostasis. See
[0165] In agreement with the correction of the lysosomal pathology, all signs of inflammation disappeared from the brains of treated MPSII mice. Four months post treatment, the signal intensities for the stainings used to detect astrocytosis (GFAP) and microgliosis (BSI-B4) were similar in treated MPSII mice and in healthy animals in different brain regions, as opposed to the signal documented in MPSII mice administered with the control Null AAV9 vector that showed a clear upregulation of these markers of neuroinflammation. See
[0166] AAV9 vectors administered to the CSF leak to the periphery and transduce the liver. See Haurigot et al., Clin Invest. 2013; 123(8):3254-3271, Ribera et al., Hum Mol Genet. 2014; doi: 10.1093/hmg/ddu727. Accordingly, an increase in Iduronate-2-sulfatase activity was documented 4 months after gene transfer in the liver and serum of MPSII mice treated with AAV9-CAG-omIDS, reaching levels of approximately 1700% and 700% of the levels observed in healthy animals, respectively. See
[0167] Four months after treatment IDS activity was also increased in lung and was particularly high in heart. See
[0168] In agreement with the GAG content data, the weight of the liver was normalized in 6-month-old MPSII mice treated with AAV9-CAG-omIDS at the age of 2 months. See
[0169] The impact of the intra-CSF administration of AAV9-CAG-omIDS on behaviour was assessed at 6 months of age with the Open Field test, which evaluates the general locomotor and exploratory activity of mice in unknown surroundings. Untreated and AAV9-null-treated MPSII mice displayed reduced exploratory activity compared with healthy mice in terms of the time spent in the centre and in the periphery, the number of entries in the centre and in the periphery and the total number of fast movements. Intracisternal administration of AAV9-CAG-omIDS completely corrected behavioural deficits in MPSII mice. See
[0170] Finally, the therapeutic efficacy of intra-CSF AAV9-CAG-omIDS treatment was evaluated by comparing the survival of untreated and treated MPSII mice. AAV9-CAG-omIDS gene therapy considerably extended the lifespan of MPSII mice. See
Example 14: Intracisternal Delivery of Different Doses of AAV9-CAG-omIDS: Dose-Response Study
[0171] Four different doses (1.5810.sup.9, 510.sup.9, 1.5810.sup.10 and 510.sup.10 vg/mouse) of AAV9-CAG-omIDS vectors were administered to 2-month-old MPSII animals via intracisternal injection in a total volume of 5 p1.
[0172] One and a half months post vector administration, animals were sacrificed and tissues harvested. Iduronat-2-sulfatase activity was measured in different parts of the brain (sections I-V). Activity increased with dose and ranged from 0.8 and 53% of WT activity in the different regions. See
[0173] In the liver and serum, activity also increased with dose, ranging from 20% to 4300% in the liver and 0.4% and 1100% in serum. No IDS activity was detectable in serum with the 2 lowest doses (1.5810.sup.9, 510.sup.9 vg/mouse). See