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WO2016112350A1 - Procédés et compositions pour le diagnostic et le traitement de troubles du stockage lysosomal - Google Patents

Procédés et compositions pour le diagnostic et le traitement de troubles du stockage lysosomal Download PDF

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Publication number
WO2016112350A1
WO2016112350A1 PCT/US2016/012747 US2016012747W WO2016112350A1 WO 2016112350 A1 WO2016112350 A1 WO 2016112350A1 US 2016012747 W US2016012747 W US 2016012747W WO 2016112350 A1 WO2016112350 A1 WO 2016112350A1
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lysosomal storage
nucleic acid
gene
mutation
backbone
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Lijun Xia
Klaas Wierenga
Patrick M. Gaffney
Jianxin Fu
Yuji Kondo
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Oklahoma Medical Research Foundation
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Oklahoma Medical Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21112—Site-1 protease (3.4.21.112), i.e. subtilisin kexin isozyme-1

Definitions

  • the lysosome is an essential cell organelle that breaks down unwanted biomolecules, including proteins, nucleic acids, carbohydrates, and lipids.
  • the lysosome degrades these unwanted materials via lysosomal hydrolases, which are synthesized in the endoplasmic reticulum (ER) and then transported to the Golgi apparatus.
  • ER endoplasmic reticulum
  • these lysosomal enzymes are specifically tagged by mannose-6-phosphate (M6P) to be targeted to the lysosome. Mutations in genes controlling this machinery cause accumulation of substances in lysosomes and defective cellular functions. Collectively, these diseases are called lysosome storage diseases (LSDs).
  • Mucolipidosis type II (ML-II) and III (ML-III) are autosomal recessive genetic LSDs, which are characterized by accumulation of mucosaccharides and mucolipids in lysosomes, caused by loss of function mutations in N-acetylglucosamine-1 (GlcNAc-1)- phosphate transferase ⁇ , ⁇ (G PTAB) and N-acetylglucosamine-1 -phosphate transferase ⁇ (GNPTG).
  • GlcNAc-1 N-acetylglucosamine-1
  • G PTAB N-acetylglucosamine-1 -phosphate transferase ⁇
  • GNPTG N-acetylglucosamine-1 -phosphate transferase ⁇
  • GNPTAB/ GNPTG encode GlcNAc-1 -phosphotransferases required for M6P modifications.
  • the present disclosure relates to new diagnostic, research and therapeutic applications for a lysosomal storage disorder such as ML-III or related disorders presenting with similar phenotypes. These new applications are based on the discovery of novel compound heterozygotic mutations (missense and nonsense) in the membrane-bound transcription factor peptidase site 1 (MBTPS1) gene associated with a phenotype related to ML-III.
  • MTPS1 membrane-bound transcription factor peptidase site 1
  • MBTPS1 encodes a membrane-bound enzyme called site-1 protease (SIP), which is a member of serine proteases that act as a proprotein convertase responsible for the cleavage of several precursor substrates such as sterol regulatory element-binding proteins (proSREBPs), proSIP, and proG PTAB.
  • SIP site-1 protease
  • proSREBPs sterol regulatory element-binding proteins
  • proSIP proSIP
  • proG PTAB proG PTAB
  • labeled nucleic acid probes that specifically hybridize to portions of the MB TPS 1 gene harboring the mutations are provided.
  • methods for identifying the presence of a MBTPS1 mutation or its gene product for diagnosing a lysosomal storage disorder are provided.
  • methods for screening therapeutic options for a lysosomal storage disorder in cells transformed with the mutated MBTPS1 gene are provided.
  • compositions and methods involving the use of gene therapy to replace the mutated form of the MBTPS1 gene with the non- mutated form of the MBTPS1 gene enzyme replacement therapy, other peptide-based therapeutic approaches, and antisense morpholino oligonucleotides to prevent the production of nonfunctional splicing variant SIP proteins are provided.
  • a portion of exon 9 of a pre-mRNA transcript of the MB TPS 1 gene with a cryptic splicing site corresponds to SEQ ID NO: 1.
  • a portion of intron 8 and exon 9 of a pre- mRNA transcript of the MB TPS 1 gene that encompasses the splicing acceptor site corresponds to SEQ ID NO: 2.
  • the splice variant form of the mRNA transcript of the MBTPS1 gene corresponding to a 41 base pair deletion in exon 9 corresponds to SEQ ID NO: 3.
  • a cDNA counterpart of the mRNA transcript of SEQ ID NO: 3 corresponds to SEQ ID NO: 4.
  • the amino acid sequence of the splice variant SIP protein corresponding to the 41 base pair deletion in exon 9 corresponds to SEQ ID NO: 5.
  • SEQ ID NO: 6 corresponds to a portion of the nucleotide sequence of the splice variant mRNA of SEQ ID NO: 3.
  • the nucleotide sequence of SEQ ID NO: 7 corresponds to a portion of the MBTPS1 gene containing the nucleotide substitution of an adenine with a guanine at position 84, 121,003 of chromosome 16.
  • the amino acid sequence of wild-type, full length SIP protein corresponds to SEQ ID NO: 8.
  • the mRNA transcript coding for the protein of SEQ ID NO: 8 corresponds to SEQ ID NO: 9.
  • the cDNA sequence that is complementary to the mRNA sequence of SEQ ID NO: 9 corresponds to SEQ ID NO: 10.
  • the lysosomal storage disorder is the result of a mutation in the MB TPS 1 gene. In certain embodiments, the lysosomal storage disorder is the result of a mutation in the MB TPS 1 gene which comprises insertion of an adenine at position 84,132,793 of chromosome 16 or substitution of an adenine with a guanine at position 84, 121,003 of chromosome 16.
  • a synthetic oligonucleotide comprising a nucleotide sequence of at least 18 consecutive nucleotides, wherein the nucleotide sequence is complementary to a target sequence on a pre-messenger RNA (pre-mRNA) transcript of the MB TPS 1 gene, and wherein the target sequence includes a cryptic splicing site resulting from a mutation in the MB TPS 1 gene is provided.
  • pre-mRNA pre-messenger RNA
  • the cryptic splicing site is located in exon 9 of the pre-mRNA transcript of the MBTPS1 gene.
  • the mutation in the MBTPS1 gene is located at position 84,121,003 of chromosome 16.
  • a synthetic oligonucleotide comprising a nucleotide sequence of at least 18 consecutive nucleotides, wherein the nucleotide sequence is complementary to a target sequence on a pre-messenger RNA (pre-mRNA) transcript of the MB TP SI gene, and wherein the target sequence includes a splicing acceptor site at the 3' end of intron 8 is provided.
  • pre-mRNA pre-messenger RNA
  • the target sequence spans the junction of intron 8 and exon 9.
  • a method for treating a lysosomal storage disorder in a patient comprises administering a synthetic oligonucleotide as described above to said patient.
  • a method for treating a lysosomal storage disorder in a patient comprises administering an antisense morpholino oligonucleotide to said patient, wherein the antisense morpholino oligonucleotide comprises a nucleotide sequence of at least 18 consecutive nucleotides, wherein the nucleotide sequence is complementary to a target sequence on a pre-messenger RNA (pre-mRNA) transcript of the MBTPS1 gene, and wherein the target sequence comprises SEQ ID NO: 1 (uugaagguaacauc) or a portion of SEQ ID NO: 1, wherein the portion includes the third guanine base reading from 5' to 3' .
  • pre-mRNA pre-messenger RNA
  • a method for treating a lysosomal storage disorder in a patient comprises administering an antisense morpholino oligonucleotide to said patient, wherein the antisense morpholino oligonucleotide comprises a nucleotide sequence of at least 18 consecutive nucleotides, wherein the nucleotide sequence is complementary to a target sequence on a pre-messenger RNA (pre- mRNA) transcript of the MB TPS 1 gene, and wherein the target sequence comprises SEQ ID NO: 2 (uuggucucaauauagcacucugaauaaccc) or a portion thereof that includes the adenine- guanine dinucleotide sequence.
  • pre- mRNA pre-messenger RNA
  • a method for treating a lysosomal storage disorder comprises administering to a subject suffering from the lysosomal storage disorder a peptide having a sequence corresponding to at least a portion of SIP. In other embodiments, a method for treating a lysosomal storage disorder comprises administering to a subject suffering from the lysosomal storage disorder a nucleic acid molecule coding for at least a portion of a SIP protein.
  • the nucleic acid molecule is DNA, wherein the sequence of the DNA encodes a SIP polypeptide having the amino acid sequence of SEQ ID NO: 8; and wherein the nucleic acid molecule further comprises a promoter sequence sufficient for inducing transcription of the DNA in a target cell.
  • a complementary DNA (cDNA) molecule which comprises a sequence based on an expressed mRNA of a splice variant form of the human MB TPS 1 gene, wherein the splice variant form of the human MBTPS1 gene is a 41 base pair deletion of exon 9 and comprises SEQ ID NO: 3.
  • a complementary DNA (cDNA) molecule comprises a sequence based on an expressed mRNA of a splice variant form of the human MBTPSl gene, wherein the splice variant form of the human MBTPSl gene does not include exon 9.
  • a complementary DNA (cDNA) molecule havening a sequence based on the expressed mRNA of the human MBTPSl gene carrying a mutation associated with a lysosomal storage disorder is provided.
  • an antibody capable of binding a protein coded by an expressed mRNA of a splice variant form of the human MBTPSl gene, wherein the splice variant form of the human MBTPSl gene is a 41 base pair deletion of exon 9 is provided.
  • a monoclonal or polyclonal antibody to a human peptide expressed from the MBTPSl gene carrying a mutation is provided.
  • the expressed mRNA of the splice variant form of the human MBTPSl gene comprises SEQ ID NO: 3.
  • a method of diagnosing a lysosomal storage disorder comprises the following steps: (a) obtaining a biological sample from a subject suspected of having a lysosomal storage disorder; (b) isolating the mRNA from the biological sample; (c) converting the mRNA to cDNA; (d) performing a polymerase chain reaction to generate an amplified portion of cDNA sufficient to determine if a deletion mutation is present in exon 9 of the human MBTPSl gene; and (e) determining the presence or absence of the deletion mutation by the number of nucleotides in the amplified portion, or isolating and sequencing the amplified portion, wherein presence of the deletion mutation is indicative of the lysosomal storage disorder.
  • a method for diagnosing a lysosomal storage disorder comprises obtaining a nucleic acid sample from a subject suspected of having the lysosomal storage disorder, applying a nucleic acid probe to the nucleic acid sample, the nucleic acid probe comprising an oligonucleotide of from about 8 to about 1000 nucleotides in length, wherein the sequence of the oligonucleotide is complementary to a target sequence, wherein the target sequence comprises a mutation in the MBTPSl gene associated with the lysosomal storage disorder, and a label attached to the oligonucleotide, and detecting the presence or absence of the label thereby indicating hybridization of the nucleic acid probe to a DNA or RNA molecule in the nucleic acid
  • a method for diagnosing a lysosomal storage disorder comprises introducing an antibody to a biological sample from a subject suspected of having the lysosomal storage disorder, wherein the antibody is specific for a mutated SIP peptide associated with the lysosomal storage disorder.
  • a nucleic acid probe comprises an oligonucleotide of from about 8 to about 1000 nucleotides in length, wherein the sequence of the oligonucleotide is complementary to a target sequence, wherein the target sequence comprises a mutation in the MB TPS 1 gene and a label attached to the oligonucleotide.
  • the mutation is located at position 84,132,793 of chromosome 16.
  • the mutation at position 84,132,793 of chromosome 16 comprises insertion of a nucleotide.
  • the nucleotide is adenine.
  • the mutation is located at position 84, 121, 003 of chromosome 16. In some embodiments the mutation at position 84, 121,003 of chromosome 16 comprises substitution of an adenine with a guanine. In an embodiment, the nucleotide is adenine. In some embodiments the target sequence comprises SEQ ID NO: 3. In other embodiments, the target sequence comprises SEQ ID NO: 1 (uugaagguaacauc) or a portion of SEQ ID NO: 1, wherein the portion includes the third guanine base reading from 5' to 3' . In some embodiments the target sequence comprises SEQ ID NO: 7 or a portion of SEQ ID NO: 7, wherein the portion includes the third guanine base reading from 5' to 3' .
  • a method for screening potential therapeutics for a lysosomal storage disorder comprises culturing a transformed eukaryotic host cell containing an altered MBTPS1 gene causing translation of a mutated SIP or preventing translation of SIP wherein such mutated SIP is associated with the lysosomal storage disorder, observing a phenotype related to the lysosomal storage disorder, applying a composition suspected of being a therapeutic for the lysosomal storage disorder to the transformed eukaryotic host cell, and observing the effect of the composition on the phenotype, wherein a degree of reduction in the phenotype in the presence of said compound is indicative of a lysosomal storage disorder therapeutic.
  • the mutation comprises insertion of a nucleotide at either position 84, 132,793 of chromosome 16 or a substitution of a nucleotide at position 84, 121,003 of chromosome 16.
  • the nucleotide is adenine.
  • the mutation comprises insertion of an adenine at position 84, 132,793 of chromosome 16 or substitution of an adenine with a guanine at position 84, 121,003 of chromosome 16.
  • the mutated SIP arises from a deletion mutation in exon 9 of the MBTPS1 gene.
  • the mutated SIP comprises SEQ ID NO: 5.
  • a composition comprising a purified human recombinant SIP protein comprising all or a portion of the amino acid sequence for SIP is provided.
  • a pharmaceutical composition comprising a purified human recombinant SIP protein comprising all or a portion of the amino acid sequence for SIP and a pharmaceutically acceptable carrier is provided.
  • an enzyme replacement therapy comprises a purified human recombinant SIP protein comprising all or a portion of the amino acid sequence of SEQ ID NO: 8.
  • a method for treating a sybject with a lysosomal storage disorder comprises administering the enzyme replacement therapy to the subject.
  • FIG. 1 provides exome sequence analysis of the father and female offspring subject thereof (proband) of Example 1 demonstrating the location of an inherited mutation (insertion of adenine) in the MB TPS 1 gene at position 84, 132,793 of chromosome 16 changing an aspartic acid residue at amino acid (AA) position 96 to a premature stop codon (D96*) in the MBTPS1 gene product (SIP).
  • FIG. 1 discloses SEQ ID NO: 11.
  • FIG. 2 provides exome sequence analysis of the mother and the proband of Example 1 demonstrating the location of an inherited mutation (a substitution of an adenine with a guanine) in the MBTPS1 gene at position 84, 121,003 of chromosome 16 changing an aspartic acid residue at amino acid (AA) position 365 to a glycine (D365G) in SIP.
  • FIG. 2 discloses SEQ ID NO: 12.
  • FIG. 3a provides a Western Blot showing overexpression of each SIP and its auto-cleavage in HEK293T cells detected with anti-FLAG antibody.
  • FIG. 3b provides a Western Blot demonstrating cleavage of proG PTAB in SRD-12B CHO cells transfected with each SIP construct detected with anti-Myc antibody.
  • S1PS414A was used as a loss of function mutation of protease activity.
  • FIG. 3B discloses "Hisx6" as SEQ ID NO: 13.
  • FIG. 4 provides a Western Blot demonstrating M6P modification in parent and patient-derived B cell lysates detected using anti-M6P antibody.
  • FIG. 5 depicts the predicted structure of human SIP.
  • the three amino acids labeled H249, D218 and S414 are the catalytic triad.
  • D365 is far from catalytic triad.
  • FIG. 6 provides a histogram representing mRNA levels of MBTPS1MBTPS2, GNPTAB and GNPTG in immortalized B cells taken from the patient and her parents described in Example 1.
  • FIG. 7 is a representative therapeutic strategy using antisense morpholino oligonucleotides to avoid the cryptic pathogenic spicing donor (SD) in Exon 9 of the MBTPSl gene.
  • FIG. 7 discloses "UUGAAGGUAAC AUC " as SEQ ID NO: 1.
  • FIG. 8 is a representative therapeutic strategy using antisense morpholino oligonucleotides to block the splicing acceptor site (SA) of Intron 8.
  • SA splicing acceptor site
  • FIG. 9 identifies the mother-derived mutation (D365G) that yields a cryptic splicing site in exon 9 of the pre-RNA transcript (referred to as "Primary RNA transcript") of the MBTPSl gene and the resulting mRNA transcript which includes a 41 base pair deletion of exon 9.
  • D365G mother-derived mutation
  • Primary RNA transcript a cryptic splicing site in exon 9 of the pre-RNA transcript (referred to as "Primary RNA transcript") of the MBTPSl gene and the resulting mRNA transcript which includes a 41 base pair deletion of exon 9.
  • lysosomal storage disorder means a disorder caused by lysosomal dysfunction usually as a consequence of deficiency of an enzyme(s) required for the metabolism of lipids, glycoproteins or mucopolysaccharides, such as mucolipidosis type III, a related disorder displaying a similar phenotype and other lysosomal storage diseases.
  • mutation in the MB TPS 1 gene means any mutation in the MB TPS 1 gene, whether in an exon, intron, promoter, enhancer, 3' flanking region, or other regulatory, non-coding region.
  • the mutation associated with this phrase may further encompass all forms of mutations including deletions, insertions and point mutations in the coding or non-coding nucleic acid sequence as well as epigenetic modifications to the coding or non-coding sequence or associated proteins (e.g. histones) such as the presence or absence of methylation, phosphorylation, or acetylation.
  • Deletions may be of the entire gene or of only a portion of the gene.
  • Point mutations may result in stop codons, alternative splice variants, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the disease tissue, and are not inherited in the germline.
  • Germline mutations can be found in any of a body's tissues and are inherited. Both somatic and germline mutations are included in this definition. This phrase should also be understood to encompass corresponding mutations in RNA transcribed from the various described mutations in the MB TPS 1 gene and the cDNA produced from such RNA. Specific examples of mutations in the MB TPS 1 gene include insertion of a nucleotide (including, but not limited to adenine) in the MB TPS 1 gene at position 84,132,793 of chromosome 16, and substitution of one nucleotide for another (e.g. adenine fwith guanine) at position 84, 121,003 of chromosome 16.
  • a nucleotide including, but not limited to adenine
  • substitution of one nucleotide for another e.g. adenine fwith guanine
  • a therapeutic composition is provided that is capable of binding to a MB TPS 1 pre-mRNA transcript thereby modulating splicing and restoring or enhancing the function of the MBTPS1 gene product.
  • the present invention thus identifies sequences within the MBTPS1 pre-mRNA which are targeted in order to modulate the splicing cascade of the MB TPS 1 pre-mRNA transcript.
  • the sequences include those of exon 9 that correspond to the adenine to guanine mutation at position 84,121,003 of chromosome 16.
  • the sequence in exon 9 that is targeted by the therapeutic composition comprises SEQ ID NO: 1 (uugaagguaacauc) or a portion thereof, wherein any portion of SEQ ID NO: 1 includes the third guanine base reading from 5' to 3' as shown in FIGS. 7 and 9.
  • the sequences include those encompassing the splicing acceptor site for the intron8-exon 9 junction of the MBTPSl pre-mRNA transcript as shown in FIG. 8.
  • sequence that encompasses the splicing acceptor site includes sequence from intron 8 and exon 9 and comprises SEQ ID NO: 2 (uuggucucaauauagcacucugaauaaccc) or a portion thereof, wherein any portion thereof includes the adenine-guanine dinucleotide sequence of SEQ ID NO: 2.
  • the therapeutic composition comprises a synthetic oligonucleotide molecule, wherein the synthetic oligonucleotide comprises a nucleotide sequence of at least 18 consecutive nucleotide bases that is complementary to a target sequence in exon 9 of a pre-mRNA transcript of the MBTPS1 gene, and suppressing the cryptic splicing site therein generated by the adenine to guanine mutation at position 84,121,003 of chromosome 16.
  • the mutation at position 84, 121,003 of chromosome 16 comprises substitution of an adenine with a guanine.
  • the target sequence comprises SEQ ID NO: 1 (uugaagguaacauc) or a portion of SEQ ID NO: 1, wherein the portion includes the third guanine base reading from 5' to 3'.
  • the target sequence comprises SEQ ID NO: 2 (uuggucucaauauagcacucugaauaaccc) or a portion thereof that includes the adenine-guanine dinucleotide sequence.
  • the consecutive nucleotide bases are linked by a backbone selected from the group consisting of a phosphate-ribose backbone, a phosphate- deoxyribose backbone, a 2'-0-methyl-phosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, an alternating locked nucleic acid backbone and a phosphorothioate backbone.
  • a backbone selected from the group consisting of a phosphate-ribose backbone, a phosphate- deoxyribose backbone, a 2'-0-methyl-phosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, an alternating locked nucleic acid backbone and
  • the nucleotide sequence comprises at least 18 nucleotides and more generally from about 18 or at least 25 nucleotides. In other certain embodiments, the nucleotide sequence comprises about 20 to 30 nucleotides, or from about 20 to 28, about 20 to 26 or about 22 to 26 nucleotides. In specific embodiments, the nucleotide sequence comprises 22, 23, 24, 25, or 26 nucleotides. Each possibility represents a separate embodiment of the present disclosure.
  • the synthetic oligonucleotide is an antisense morpholino oligonucleotide.
  • the synthetic oligonucleotide nucleotide sequence comprises 20 to 25 consecutive nucleotides.
  • the therapeutic composition comprises a morpholino oligonucleotide comprising a sequence sufficient to form a complex with a region of exon 9 and/or intron 8 in the MBTPSl pre-mRNA transcript.
  • the region of exon 9 and/or intron 8 can include any sequence that signals a cryptic splicing site including, but not limited to the site that corresponds to the substitution of adenine with guanine at position 84, 121,003 in chromosome 16 and SEQ ID NO: 1, and SEQ ID NO: 2.
  • the morpholino oligonucleotide may include an antisense oligonucleotide comprised of morpholino subunits and phosphorous-containing intersubunit linkages comprising a sequence of at least 12 contiguous base-pairing moieties complementary to various sequences of exon 9 of the MBTPSl pre-mRNA transcript and sequences in intron 8 thereof, or in sequences spanning intron 9 and exon 9 thereof, and wherein the antisense oligonucleotide is capable of binding to the target pre-mRNA to form a heteroduplex structure.
  • the phosphorous-containing intersubunit linkages can be phosphorodiamidate.
  • the therapeutic compositions may comprise other antisense oligonucleotides such as 2'-0-methyl-phosphorothioate (20MP), peptide nucleic acids (PNAs), 2-methoxy ethyl phosphorothioate (MOE) and alternating locked nucleic acids (LNAs).
  • MP 2'-0-methyl-phosphorothioate
  • PNAs peptide nucleic acids
  • MOE 2-methoxy ethyl phosphorothioate
  • LNAs alternating locked nucleic acids
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a synthetic oligonucleotide molecule as described in any of the embodiments above, wherein the synthetic oligonucleotide is formulated as a pharmaceutical composition and further comprising a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers and formulations for nucleic acids are discussed below in further detail.
  • the pharmaceutical composition is formulated for oral, nasal, aerosol, inhalational, abdominal, subcutaneous, intraperitoneal or intravenous administration.
  • a complementary DNA (cDNA) molecule having a sequence based on the expressed mRNA of a splice variant form of the human MBTPSl gene is provided.
  • the splice variant form comprises a 41 base pair deletion of exon 9.
  • an antibody capable of binding a protein coded by an expressed mRNA of a splice variant form of the human MBTPSl gene is provided.
  • the splice variant form comprises a 41 base pair deletion of exon 9.
  • the present disclosure also provides methods of treating lysosomal storage disorders.
  • the method comprises administering to a patient diagnosed with a lysosomal storage disorder a composition comprising the synthetic oligonucleotide molecules described above, including antisense morpholino oligonucleotides sufficient to bind the MBTPSl pre-mRNA transcript and prevent improper splicing events as described above.
  • the lysosomal storage disorder in some embodiments, is Mucolipidosis type II or Mucolipidosis type III. In some embodiments, the lysosomal storage disorder is Mucolipidosis type II or Mucolipidosis type III or a related disorder. In other embodiments, the lysosomal storage disorder is Mucolipidosis type III. In other embodiments, the lysosomal storage disorder is Mucolipidosis type III or a related disorder.
  • the present disclosure also provides methods of diagnosing lysosomal storage disorders such as Mucolipidosis type II or type III.
  • the method comprises the following steps: (a) obtaining a biological sample from a subject suspected of having a lysosomal storage disorder; (b) isolating the RNA from the biological sample; and (c) determining the presence of a MBTPSl mutant mRNA transcript, wherein presence of the transcript is indicative of disease.
  • the MBTPSl mutant mRNA transcript includes a splice variant that is missing 41 base pairs of exon 9.
  • Determining the presence of the mutant mRNA transcript can be performed by reverse transcription of mRNA isolated from the subject to yield cDNA and then performing PCR using primers that flank the suspected alternative splicing site. The PCR products can then be loaded into agarose gels for electrophoresis to identify the presence of smaller sized products (corresponding to the deletion). Labeled probes comprising sequences specific to the splice variants of the MBTPSl mRNA transcript can also be employed for diagnostic purposes. Thus, in one embodiment, a probe is provided comprising a sequence complementary to SEQ ID NO: 6 (GACUUUGAAGGAGCUACCAGGAGG) or a portion thereof.
  • the probe must contain a sequence which is complementary to a portion of SEQ ID NO: 6 which spans the alternative splicing site yielding the mutant MBTPSl mRNA (spanning exon 9-exon 10 junction in mutant— includes the third and fourth guanine reading 5' to 3').
  • a nucleic acid probe for identification of a mutation in or around the MBTPS1 gene.
  • the nucleic acid probe may comprise an oligonucleotide of from about 8 to about 1000 nucleotides in length, wherein the sequence of the oligonucleotide corresponds to a mutation in the MBTPS1 gene associated with a lysosomal storage disorder.
  • the probe further comprises a label attached thereto for confirming hybridization of the probe to a target sequence in a sample.
  • a probe comprising a sequence complementary to SEQ ID NO: 7 (TTGAAGGTAACATC) or a portion thereof which spans the site of the substitution of adenine with guanine at position 84,121,003 of chromosome 16.
  • a probe is provided comprising a sequence complementary to SEQ ID NO; 6.
  • the probe can be designed by any method known in the art that will allow selective hybridization to a target (disease-associated) sequence having a mutation in the MB TPS 1 gene associated with a lysosomal storage disorder.
  • a target (disease-associated) sequence having a mutation in the MB TPS 1 gene associated with a lysosomal storage disorder.
  • such mutation in the target sequence may comprise only a single nucleotide difference at a particular location with respect to the wild-type (not disease-associated) sequence. This is important as the mutation may be present in only a particular tissue or cell type such that a biological sample may contain alleles that do not carry the mutation.
  • the probe may comprise any configuration known in the art necessary to confer such ability to differentiate a single nucleotide difference between a target sequence and a wild-type sequence at a particular position within a common gene locus.
  • the probe may be single-stranded or double stranded, may comprise multiple domains, some of which are not specific to the target sequence, and may include various secondary structures such as hairpin loops.
  • the probe may be designed to specifically detect single nucleotide mutations in DNA, complementary DNA (cDNA), or RNA.
  • the label attached to the oligonucleotide of the probe may include, but is not limited to fluorescent agents, biotin, radionuclides, chemiluminescent agents, enzymes, substrates, cofactors, magnetic particles and other components known in the art.
  • the probe may be double-stranded wherein one oligonucleotide strand comprises a label and the other strand a quencher specific for that label such that upon hybridization to the target sequence, the strand carrying the quencher is released thereby permitting detection of the label.
  • Other modifications to the oligonucleotide of the probe can be made to create a label such as methylation or nucleotide analog.
  • the nucleic acid probes of the present disclosure can be used in a number of established assays for diagnostic or research purposes which will be readily apparent to one of skill in the art.
  • the probe can be used to detect the presence of the mutation in a sample comprising MB TPS 1 PCR amplification products generated from a subject's DNA.
  • the probe may also be used in biopsied tissue sample or tissue slices for in situ hybridization (in the instance the mutation is expressed in the mRNA).
  • the present disclosure provides a method for diagnosing a lysosomal storage disorder using the nucleic acid probes of the present disclosure.
  • the method comprises the following steps: (a) obtaining a nucleic acid sample from a subject suspected of having the lysosomal storage disorder; (b) applying a nucleic acid probe, such as the nucleic acid probe described herein, to the nucleic acid sample; and (c) detecting the presence or absence of the label thereby indicating hybridization of the nucleic acid probe to a DNA molecule in the nucleic acid sample or lack thereof, wherein hybridization equates to the presence of the mutation of the MBTPS1 gene in the nucleic acid sample.
  • the nucleic acid sample can be obtained by isolating the genomic DNA or RNA from a biological sample of a subject. DNA extraction from blood or tissues samples is well known in the art and many extraction kits are commercially available for this purpose. In some instances, however, the nucleic acid sample may not necessarily be extracted from the biological sample such that the nucleic acid sample is maintained in the biological sample taken from the subject without isolation of the nucleic acid therein. In these instances, the probe may be applied directly to the biological sample, whether fluid or tissue.
  • detection of a mutation in the MBTPS1 gene comprises analysis of DNA sequence.
  • the method comprises the following steps: (a) obtaining a nucleic acid sample from a subject suspected of having the lysosomal storage disorder; (b) isolating a portion of the MB TPS 1 gene corresponding to a mutation associated with the lysosomal storage disorder; (c) sequencing the portion to obtain a subject sequence; and (d) identifying the presence of the mutation in the subject sequence.
  • the nucleic acid sample will often comprise isolated genomic DNA in an appropriate buffer.
  • isolation of genomic DNA from a biologic sample is well known in the art.
  • the portion of the MBTPS1 gene containing one or more mutations will typically be amplified by polymerase chain reaction (PCR) by using appropriate primers flanking the desired region of the gene.
  • PCR polymerase chain reaction
  • the target amplified region can then be isolated by a number of techniques known in the art and then subjected to direct sequencing techniques or cloned into an appropriate plasmid for sequencing.
  • RNA from a subject sample it may be desirable to isolate the RNA from a subject sample and then synthesize cDNA therefrom for sequencing.
  • the presence of the mutation in the subject sequence can be performed by determining the identity of the nucleotide at the mutation site.
  • this step can be automated wherein a specific computer program compares the subject sequence to a corresponding wild-type sequence to determine if a nucleotide insertion or substitution corresponding to the known mutation is present. This step may also be performed by manually reviewing the sequence data.
  • a method of diagnosing a lysosomal storage disorder comprises measuring the level MB TPS 1 gene product in a sample.
  • the mutations will be those leading to decreased expression of the MBTPS1 gene product.
  • mutations leading to non-functional gene products would also lead to a disease state.
  • Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the MBTPS1 gene product, or to a decrease in mRNA stability or translation efficiency. Alteration of MB TPS 1 mRNA expression can be detected by any techniques known in the art.
  • alteration of the wild-type MB TPS 1 gene can also be detected by screening for alteration of wild-type MBTPS1 protein.
  • monoclonal antibodies immunoreactive with MBTPS1 can be used to screen a sample for potential disease state. Lack of cognate antigen would indicate a MBTPS1 mutation.
  • Antibodies specific for products of mutant alleles could also be used to detect mutant MB TPS 1 gene product.
  • immunological assays can be done in any convenient formats known in the art. These include, but are not limited to Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered MB TPS 1 protein can be used to detect alteration of wild-type MBTPS1 genes. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect MBTPS1 biochemical function.
  • the present disclosure provides a non- human monoclonal or polyclonal antibody to a human peptide resulting from expression of a mutation in the MBTPS1 gene, wherein the mutation is associated with a lysosomal storage disorder.
  • antibody encompasses polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the MBTPSl polypeptides and fragments thereof or to polynucleotide sequences from the MBTPSl region, particularly from the MBTPSl locus or a portion thereof.
  • antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Monoclonal antibodies may be made by injecting mice or some other animal with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies can be screened by ELISA and tested for specific immunoreactivity with MBTPSl polypeptide or fragments thereof. These antibodies may be useful in assays as well as pharmaceuticals.
  • the present disclosure provides novel therapeutic approaches to the treatment of one or more disorders, including lysosomal storage disorders such as mucolipidosis type III or related disorders that involve mutations in the MBTPSl gene leading to decreased expression of gene products or non-functional/semi- functional gene products.
  • lysosomal storage disorders such as mucolipidosis type III or related disorders that involve mutations in the MBTPSl gene leading to decreased expression of gene products or non-functional/semi- functional gene products.
  • a purified, recombinant SIP protein, or a peptide or peptide mimetic comprising an amino acid sequence corresponding, at least in part, to the amino acid sequence of SIP is provided.
  • the peptide mimetic may comprise only a portion of the full peptide sequence or substantially all of the peptide sequence of SIP.
  • the peptide mimetic may comprise additional domains or amino acid sequences that are not naturally-occurring.
  • the peptide can be one that may not have sequence homology with SIP, but possesses SIP -like activity.
  • Other aliphatic amino acids are alanine, valine and isoleucine. It is also specifically contemplated that chemically similar amino acids may be added to one or both ends of a peptide of the invention without negatively affecting the activity of the peptide.
  • SIP protein can be used for treatment, such as enzyme replacement therapy (ERT), of subjects with lysosomal storage disorders.
  • ERT enzyme replacement therapy
  • the SIP protein (enzyme) is intravenously infused and taken up into the target cell through membrane receptors, such as mannose-6-phosphate receptor, and thereby replaces or supplements the activity of the endogenous mutated or under-expressed enzyme.
  • an enzyme replacement therapy composition comprising SIP protein
  • the SIP protein comprises purified SIP protein.
  • purified refers to proteins where various sugars or other naturally occurring post-translational modifications that interfere with the ability of the protein to recognized receptors on cells targeted for ERT are no longer present. Purification processes of this type are generally known in the art.
  • the composition comprises a purified human recombinant SIP protein comprising all or a portion of the amino acid sequence for SIP.
  • the purified human recombinant SIP includes a high mannose SIP protein, wherein "high mannose” refers to additional mannose residues and/or the presence of mannose residues that are no longer shielded by other post-translation modifications.
  • the composition comprises a purified human recombinant SIP protein comprising all or a portion of the amino acid sequence for SIP and further comprising a liquid carrier vehicle of one or more selected from the group consisting of sterile water, sodium chloride solution, and a buffer.
  • a pharmaceutical composition comprising a purified human recombinant SIP protein comprising all or a portion of the amino acid sequence for SIP and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable styrene, a pharmaceutically acceptable styrene, a pharmaceutically acceptable styrene, a pharmaceutically acceptable styrene, a pharmaceutically acceptable styl, a pharmaceutically acceptable carrier.
  • Appropriate pharmaceutical carriers and formulations are discussed in more detail below.
  • the recombinant SIP protein can be produced by obtaining cells that naturally express SIP protein and then culturing the cells under conditions sufficient to prevent removal of mannose residues or prevent post-translational modifications that interfere with the mannose residues.
  • the recombinant SIP protein can also be produced by transfecting cells with an appropriate expression vector that includes the nucleic acid coding region, such as cDNA, giving rise to the SIP protein and a promoter sufficient to provide expression of the coding region in an amount sufficient for therapeutic use.
  • the particular transfected cells do not modify the translated protein with sugars or other moieties that shield mannose residues or that prevent removal of certain mannose residues.
  • the transfected cells can be treated with a substance that inhibits mannosidase. Recombinant and other cell culture techniques such as these are described in more detail in US Patent No. 7, 138,262, which is incorporated herein by reference.
  • a method of treating a subject with a lysosomal storage disorder comprising administering a SIP enzyme replacement therapy to the subject.
  • a SIP enzyme replacement therapy comprises a purified, recombinant SIP protein having sugars or other modifications for targeting cells expressing suitable receptors in a suitable liquid vehicle carrier.
  • the step of administration comprises intravenous injection.
  • peptides, purified SIP proteins, or purified recombinant SIP proteins of the present disclosure are chemically modified.
  • peptides of the invention are acetylated at the N-terminus.
  • peptides of the invention are amidated at the C-terminus.
  • peptides of the invention are acetylated at the N-terminus and amidated at the C-terminus.
  • Peptides are acetylated or amidated by methods known in the art.
  • the peptide is glycosylated, carboxylated, phosphorylated, esterified, or converted into an acid addition salt and/or optionally dimerized, polymerized, pegylated, or otherwise conjugated.
  • the common amino acids all contain at least one chiral carbon atom. These amino acids therefore exist as pairs of stereoisomers designated as the L-isomer and the D-isomer. Most naturally occurring proteins and peptides are composed exclusively of the L-isomeric form. D-isomeric amino acids can affect the conformation of a peptide or protein and may lead to increased stability or a change in activity. These modifications may also be utilized in the peptide compositions of the present disclosure.
  • the recombinant SIP proteins of the present disclosure can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • SIP polypeptide can be extracted from SIP -producing mammalian cells.
  • the techniques of synthetic chemistry can be employed to synthesize SIP protein. Any of such techniques can provide the preparation of the present disclosure which comprises the SIP protein.. This is most readily accomplished by synthesis in a microorganism or in vitro.
  • the purified SIP proteins, purified recombinant SIP proteins, peptides, peptide fragments, or peptide mimetics of the present disclosure may be formulated for therapeutic administration in a number of different manners according to the route of administration.
  • the various protein and peptide compositions of the present disclosure may be administered by any suitable route including oral, intradermal, intraperitoneal, intranasal, subcutaneous, intramuscular, intravenous or direct injection of the composition to a tissue in need of protein replacement.
  • Formulations suitable for oral administration include, for example, solid, semi-solid and liquid systems such as, tablets; soft or hard capsules containing nano-particulates, liquids, or powders; lozenges (including liquid-filled), chews, gels, fast dispersing dosage forms, film,; ovules, extended-release, rapid-release, and sprays.
  • the peptides of the present invention are formulated for oral administration using delivery vehicles known in the art, including but not limited to, microspheres, liposomes, enteric coated dry emulsions or nanoparticles.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can contain inert diluents commonly used in the art such as, for example,
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active peptide is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cety
  • the dosage form may also comprise buffering agents.
  • the active compounds can also be in microencapsulated form with one or more excipients as noted above.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions of the present disclosure may also be used in connection with a synthetic scaffold or hydrogel applied to a target tissue.
  • a method is also provided of supplying wild- type MB TPS 1 function to a cell which carries mutant MB TPS 1 alleles.
  • the wild-type MB TPS 1 gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. More preferred is the situation where the wild-type MBTPSl gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant MBTPSl gene present in the cell. Such recombination requires a double recombination event which results in the correction of the MBTPSl gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art, and the choice of method is within the competence of the routineer.
  • Cells transformed with the wild-type MBTPSl gene can be used as model systems to study therapeutic approaches for lysosomal storage disorders.
  • the MB TPS 1 gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in target disease cells.
  • Such gene therapy is particularly appropriate for use in cells, in which the level of SIP polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given MBTPS1 gene even in those cells in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional.
  • Gene therapy would be carried out according to generally accepted methods. For example, cells from a subject would be first analyzed by the diagnostic methods described above, to ascertain the production of SIP polypeptide in the cells. A virus or plasmid vector, containing a copy of the MB TPS 1 gene linked to expression control elements and capable of replicating inside the target cells, is prepared. Suitable vectors are known in the art for this purpose. The vector is then injected into the subject, either locally or systemically. If the transfected gene is not permanently incorporated into the genome of each of the targeted cells, the treatment may have to be repeated periodically.
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods.
  • viruses have been used as gene transfer vectors, including papovaviruses, adenovirus, vaccinia virus, adeno-associated virus, herpes viruses including HSV and EBV, and retroviruses.
  • Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation, mechanical techniques such as microinjection, and membrane fusion-mediated transfer via liposomes or related nanoparticle systems where the genetic material is encapsulated into a nanoparticle, and direct DNA uptake and receptor-mediated DNA transfer.
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the target cells and not into the surrounding non-target cells.
  • the present disclosure provides a vector comprising a DNA coding for at least a portion of a SIP polypeptide, and a promoter sequence sufficient for inducing transcription of the DNA in a target cell. Additionally, the present disclosure provides, a gene therapy composition comprising the vector described above and a vehicle for delivery of the vector to the target tissue, wherein the vehicle is selected from the group consisting of a nanoparticle, a virus, a polymer-based scaffold, chemical, lipid, viral, electroporation, microinjection and ballistic.
  • the nucleic acid molecule is DNA, wherein the sequence of the DNA encodes a SIP polypeptide having the amino acid sequence of SEQ ID NO: 8; and wherein the nucleic acid molecule further comprises a promoter sequence sufficient for inducing transcription of the DNA in a target cell.
  • the nucleic acid molecule is provided in a composition comprising a nanoparticle or a virus.
  • the present disclosure provides a complementary DNA (cDNA) molecule having a sequence based on the expressed mRNA of the human MB TPS 1 gene having a mutation associated with a lysosomal storage disorder.
  • the mutation may comprise insertion of a nucleotide or substitution thereof at either position 84, 132,793 of chromosome 16 or position 84,121,003 of chromosome 16, respectively.
  • the present disclosure also provides methods for screening therapeutics in transformed cells or transgenic animals carrying a mutation in the MBTPS1 gene.
  • cells and animals which carry a mutant MBTPS1 allele can be used as model systems to study and test for substances which have potential as therapeutic agents. These may be isolated from individuals with MB TPS 1 mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the MBTPSl allele, as described above. After a test substance is applied to the cells, the transformed phenotype of the cell is determined. Any trait of transformed cells can be assessed.
  • the present disclosure provides a method for screening potential therapeutics for a lysosomal storage disorder comprising the following steps: culturing a transformed eukaryotic host cell containing an altered MBTPSl gene causing translation of a non-functional SIP, preventing translation of SIP, or preventing or decreasing expression of MBTPSl mRNA; observing a phenotype related to the lysosomal storage disorder; applying a compound suspected of being a therapeutic for the lysosomal storage disorder to the transformed eukaryotic host cell; and observing the effect of the compound on the phenotype, wherein a degree of reduction in the phenotype in the presence of said compound is indicative of a lysosomal storage disorder therapeutic.
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant MBTPSl alleles, usually from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous MBTPSl gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional methods. After test substances have been administered to the animals, the disease phenotype is assessed and compared to control animals that possess the mutation (transgenic), but did not receive the test substance. If the test substance prevents or suppresses the disease phenotype, then the test substance is a candidate therapeutic agent for the treatment of the lysosomal storage disorders identified herein. These animal models provide an extremely important testing vehicle for potential therapeutic products.
  • the second mutation was carried by the mother and was a substitution of an adenine at position chrl6: 84,121,003 to guanine, changing an aspartic acid residue at AA position 365 to a glycine residue (D365G) (Fig. 2). Both parents were heterozygotes for their respective mutations. The affected child received each mutation and is a compound heterozygote. The two unaffected siblings received only D365G mutation from the mother.
  • SIP serine proteases that act as a proprotein convertase responsible for the cleavage of several precursor substrates such as proSREBPs, proSIP, and proG PTAB.
  • GlcNAc-1 -phosphotransferase required for M6P modification is generated as an inactive form, and it needs to be cleaved by SIP to become the active form.
  • M6P modification normally occurs in ML-III patient
  • M6P modification triggers the targeting of lysosomal hydrolases from secretory pathway to the lysosomes.
  • D365G mutation generates a cryptic splicing donor site resulting in a truncated
  • transcript F father-derived mutant transcript with the premature stop codon
  • transcription M mother-derived mutant transcript without alternative splicing
  • Transcript S mother-derived mutant transcript with alternative splicing
  • RT-PCR we will determine the expression levels of these three different transcripts in patient-derived cells. Specifically, patient and parent derived B cell lines will be cultured according to standard procedures. Cells will be grown for 48 - 72 hours and total RNA will be extracted using Rneasy kit (Qiagen,CA). Reverse transcription reactions will be prepared with the iScriptTMcDNA Synthesis Kit (Invitrogen, CA).
  • PCR reactions will be performed in triplicate with SYBR Green PCR master mix (Qiagen, CA) and will be performed in an ABI 7700 sequence detector (Applied Biosy stems) according to the manufacturer's instruction. Data will be analyzed subsequently and ratios will be calculated by comparing the three different transcripts.
  • transcript M is functional
  • AMO Exon 9
  • MS mutation-generated splicing site
  • AMO antisense morpholino oligonucleotides
  • SA Intron 8 splicing acceptor
  • the 25-mer AMOs will be designed according to published articles ⁇ Stein 2001; Aartsma-Rus 2009; Chan 2006 ⁇ to target the exon 9 cryptic splicing site and acceptor site.
  • We will use scrambled AMO as a negative control, which will be used to observe whether an observed biological effect is produced by an antisense mechanism or whether it is due to a complex combination of non-sequence specific effects.
  • All of the AMOs will be synthesized and purified by Gene Tools (Philomath, OR). Endor-Portor (Gene Tools) will be used to help cells incorporate AMOs into B cells according to the manufacture's recommendations (Gene Tools; www.gene-tools.com).
  • the cell will be suspended in 5% FBS/PRMI medium 1640 and AMOs will be added directly to medium at the different concentrations. Endor-Portor (8 ⁇ /ml) will be then added to the medium and immediately mixed well. Equal volumes of Endor-Porter will be added to cell cultures with control AMOs as controls.
  • RNA will be isolated, real-time PCR will be performed as described above to determine the whether AMO specifically blocks the cryptic splicing site based on the mRNA expression in cells treated with AMOs vs cells with control oligos.
  • Western blots with an anti-SIP mAb (Marschner K) will be used to detect whether the splicing-directed therapy restores SIP function in patient-derived cells.

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Abstract

La présente invention repose sur la découverte de nouvelles mutations hétérozygotes composées d'un gène appelé site 1 de la peptidase du facteur de transcription lié à la membrane (MBTPSl, l'une est une mutation faux-sens, D365G, l'autre est une mutation non-sens). MBTPSl code pour une enzyme liée à la membrane appelée protéase de site 1 (SIP), qui fait partie de la famille des sérine protéases agissant en tant que convertase de proprotéine responsable du clivage de plusieurs substrats de précurseur, telles que les protéines se liant à l'élément régulateur du stérol (proSREBPs), proSIP et proGNPTAB. Sur la base de cette découverte, la présente invention concerne de nouvelles compositions thérapeutiques et diagnostiques ainsi que des procédés permettant d'identifier lesdites mutations et des méthodes de traitement de sujets hébergeant celles-ci, telles qu'une thérapie de remplacement enzymatique et l'utilisation d'oligonucléotides de synthèse pour bloquer l'épissage cryptique qui résulte de ces mutations, ou pour moduler d'une autre manière l'épissage afin d'éviter le site d'épissage cryptique.
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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2019084024A1 (fr) * 2017-10-24 2019-05-02 Oklahoma Medical Research Foundation Traitement de maladies du squelette provoquées par des anomalies de la circulation des protéines intracellulaires
CN111770762A (zh) * 2017-10-24 2020-10-13 俄克拉荷马医学研究基金会 对由细胞内蛋白运输缺陷引起的骨骼疾病的治疗
US12031136B2 (en) 2017-10-24 2024-07-09 Oklahoma Medical Research Foundation Treatment for skeletal diseases caused by intracellular protein trafficking defects

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