WO2012047093A1 - Traitement du syndrome de sjögren - Google Patents

Traitement du syndrome de sjögren Download PDF

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Publication number
WO2012047093A1
WO2012047093A1 PCT/NL2010/050650 NL2010050650W WO2012047093A1 WO 2012047093 A1 WO2012047093 A1 WO 2012047093A1 NL 2010050650 W NL2010050650 W NL 2010050650W WO 2012047093 A1 WO2012047093 A1 WO 2012047093A1
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Prior art keywords
baff
vector
oligonucleotide
cell
cells
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PCT/NL2010/050650
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English (en)
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Jacques-Eric Gottenberg
Xavier Mariette
Patrick A. Dreyfus
John A. Chiorini
Jelle Lucas Guido Vosters
Nienke Roescher
Margarita Jacoba Bernadetta Maria Vervoordeldonk
Paul Peter Tak
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Academisch Ziekenhuis Bij De Universiteit Van Amsterdam
Arthrogen B.V.
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Priority to PCT/NL2010/050650 priority Critical patent/WO2012047093A1/fr
Publication of WO2012047093A1 publication Critical patent/WO2012047093A1/fr

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    • 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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • the invention provides an antisense oligonucleotide able to induce the skipping of an exon of a BAFF mRNA in order to produce a truncated form of a BAFF protein when present in a cell expressing the BAFF mRNA.
  • This oligonucleotide may be used for treating the Sjogren's syndrome.
  • Sjogren' s syndrome is an innate-immune triggered autoimmune epithelitis, responsible for disabling dryness, fatigue, pain and systemic involvement.
  • the pathogenesis of the disease involves the activation of innate immunity, and interferon pathways resulting in the activation of adaptive immunity, notably B lymphocytes.
  • Targeting B cells either using monoclonal antibodies against CD20 or CD22 may result in decreased dryness. This suggests that dryness is not the only consequence of damage but also from ongoing B-cell activation, maybe through the antigen-presenting properties of B cells, their co-stimulatory effect on T cells and autoantibody secretion.
  • BAFF B-cell activating factor or BLyS
  • BAFF B-cell activating factor
  • BLyS B-cell activating factor
  • BAFF is a cytokine of the TNF family, which plays a pivotal role in the activation and survival of autoreactive B-cells.
  • BAFF transgenic mice have a phenotype of SLE and SS and levels of BAFF are increased in serum, saliva and target organs of patients with pSS, and in serum of various other autoimmune diseases and in serum and lymphoid proliferations of patients with lymphomas, notably those with a bad prognosis.
  • BAFF is not only over-expressed by myeloid and dendritic cells, and T and B lymphocytes (Daridon C, Arthritis Rheum. (2007), 56: 1134-44) but also by salivary gland epithelial cells and by epithelial cells of the ocular surface.
  • epithelial cells are induced to secrete BAFF locally, inside the target organs of autoimmunity in SS, which in turn play a pivotal role in the activation of adaptive immunity.
  • soluble receptors and monoclonal antibodies such as TACI-Fc, BAFF-R-Fc or belimumab, currently evaluated in clinical trials of patients with SLE, RA and blood malignancies, have no impact on BAFF intracellular processing and secretion.
  • the invention provides a new treatment for SS which does not have all the drawbacks of existing treatments and which induces a specific decrease of BAFF secretion.
  • BAFF secretion is to target BAFF messenger RNA (mRNA), using either small-interfering RNA or micro-RNAs, or by regulating BAFF mRNA splicing.
  • mRNA BAFF messenger RNA
  • delta-BAFF a splice variant of BAFF, with a 57-bp single exon deletion (exon 3 and 4 are skipped in human and mouse, respectively) inhibits BAFF secretion and activity by affecting the intracellular association of BAFF and its receptor binding specificity.
  • delta-BAFF prevents the intracellular binding of BAFF to other monomers of BAFF and APRIL, and inhibits the shedding of membrane-bound BAFF.
  • Full-length BAFF is naturally more abundant than delta-BAFF.
  • delta-BAFF delta-BAFF in patients with pSS.
  • an antisense oligonucleotide able to induce the skipping of an exon of a BAFF mRNA in order to produce a truncated form of a BAFF protein when present in a cell expressing the BAFF mRNA.
  • Such oligonucleotide is said functional.
  • a preferred human full length BAFF protein is represented by an amino acid sequence comprising or consisting of SEQ ID NO: l .
  • This preferred human full length BAFF protein is preferably encoded by a nucleic acid molecule represented by a nucleotide sequence comprising or consisting of SEQ ID NO:2.
  • a preferred murine full length BAFF protein is represented by an amino acid sequence comprising or consisting of SEQ ID NO:3.
  • This preferred murine full length BAFF protein is preferably encoded by a nucleic acid molecule represented by a nucleotide sequence comprising or consisting of SEQ ID NO:4.
  • a preferred human truncated BAFF protein is represented by an amino acid sequence comprising or consisting of SEQ ID NO:5.
  • This preferred human truncated BAFF protein is preferably encoded by a nucleic acid molecule represented by a nucleotide sequence comprising or consisting of SEQ ID NO:6.
  • a preferred murine truncated BAFF protein is represented by an amino acid sequence comprising or consisting of SEQ ID NO:7.
  • This preferred murine truncated BAFF protein is preferably encoded by a nucleic acid molecule represented by a nucleotide sequence comprising or consisting of SEQ ID NO:8.
  • an antisense oligonucleotide is such that in said cell less secretion of a full length BAFF protein is found compared to the secretion of the same full length BAFF protein in a cell wherein said oligonucleotide is not present.
  • This is a preferred assay for testing the functionality of the oligonucleotide of the invention.
  • oligonucleotide may also be called an antisense oligonucleotide.
  • a preferred cell is the cell line U937 or human salivary epithelial duct cells or human salivary gland cells (HSG).
  • Another cell may be a murine cell from a NOD mouse, preferably a salivary duct epithelial cells from said mouse or a cell from a treated human being.
  • a full length/truncated BAFF protein is preferably a human full length/truncated or a murine full length./truncated proteins.
  • Preferred human and murine full length/truncated BAFF proteins have already been defined herein.
  • Preferred primers are identified in the experimental part.
  • a preferred treated cell from a human being is a salivary epithelial duct cells.
  • less is at least 5% less or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% or at least 70%, or at least 80%, or at least 90%, or at least 100% as assessed by Northern blotting. Most preferably, no full length BAFF is detectable outside of the cells.
  • the assessment of the functionality of said oligonucleotide may be carried out at the mRNA level, preferably using RT-PCR or Northern blotting. Alternatively, the assessment of the functionality may be carried out at the protein level, preferably using western blot analysis or immunofluorescence analysis of cross-sections.
  • An oligonucleotide of the invention may target or may be complementary to or may bind to an exonic sequence of a BAFF mRNA.
  • An oligonucleotide as used herein preferably comprises an antisense oligonucleotide or antisense oligoribonucleotide. In a preferred embodiment an exon skipping technique is applied.
  • An antisense oligonucleotide is preferably
  • a part of BAFF mRNA to which an oligonucleotide is complementary may also be called a contiguous stretch of a BAFF mRNA.
  • a preferred exon to which an oligonucleotide of the invention is complementary to or binds is part of exon 3 of a human BAFF mRNA or part of exon 4 of a murine BAFF mRNA.
  • This oligonucleotide may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. " Part of has been defined above. Exon 3 of a human BAFF mRNA corresponds to exon 4 of a murine BAFF mRNA.
  • a preferred sequence of a nucleic acid encoding exon 3 of a human BAFF mRNA is represented by SEQ ID NO: 18.
  • a preferred sequence of a nucleic acid encoding exon 4 of a murine BAFF mRNA is represented by SEQ ID NO: 19. Therefore a preferred oligonucleotide is encoded by a nucleic acid molecule that binds or is complementary to a contiguous stretch of SEQ ID NO: 18 or SEQ ID NO: 19. This contiguous stretch may have at least 8, 10, 13, 15, 20 nucleotides.
  • said contiguous stretch may also have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides.
  • a preferred murine BAFF antisense oligonucleotide is encoded by a nucleic acid molecule represented by a sequence comprising or consisting of SEQ ID NO:9.
  • a preferred human BAFF antisense oligonucleotide is encoded by a nucleic acid molecule represented by a sequence comprising or consisting of SEQ ID NO:20.
  • complementarity is used herein to refer to a stretch of nucleic acids that can hybridise to another stretch of nucleic acids under physiological conditions.
  • Different types of nucleic acid may be used to generate an oligonucleotide.
  • said oligonucleotide comprises RNA, as RNA/RNA hybrids are very stable.
  • oligonucleotide may be modified in order to provide an additional property, for instance resistance to endonucleases, exonucleases, and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue-specificity, etc.
  • additional property for instance resistance to endonucleases, exonucleases, and RNaseH, additional hybridisation strength, increased stability (for instance in a bodily fluid), increased or decreased flexibility, reduced toxicity, increased intracellular transport, tissue- specificity, etc.
  • Dose ranges of oligonucleotide according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist.
  • An oligonucleotide as defined herein may be used at a dose which is from 0.1 to 20 mg/kg.
  • In vivo use include the use in an animal model such as a NOD mouse or in a patient.
  • a concentration of an oligonucleotide as defined herein, which is from 0.1 nM to 1 ⁇ is used.
  • this range is for in vivo use in a cellular model such as a U937 cell or a human salivary gland (HSG) cell or in salivary duct epithelial cells from a NOD mouse.
  • HSG human salivary gland
  • oligonucleotide(s) as given above are preferred concentrations or doses for in vitro or ex vivo uses.
  • concentration or dose of oligonucleotide(s) used may further vary and may need to be optimised any further.
  • An oligonucleotide as defined herein for use according to the invention may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing Sjogren's syndrome, and may be administered in vivo, ex vivo or in vitro. Said oligonucleotide may be directly or indirectly administrated to a cell, tissue and/or an organ in vivo of an individual affected by or at risk of developing the
  • a preferred tissue or organ is a salivary gland.
  • a preferred cell is a ductal epithelial cell.
  • An oligonucleotide of the invention may be indirectly administrated using suitable means known in the art.
  • An oligonucleotide may for example be provided to an individual or a cell, tissue or organ of said individual in the form of a vector or an expression vector wherein the vector or expression vector comprises a nucleic acid molecule encoding the antisense oligonucleotide.
  • the expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle.
  • a viral vector comprising an expression cassette or a transcription cassette that drives expression or transcription of the antisense oligonucleotide as identified herein.
  • a preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lenti virus vector and the like.
  • AAV adeno-associated virus vector
  • retroviral vector such as a lenti virus vector and the like.
  • vectors are delivered into the submandibular glands by retrograde ductal instillation using a thin cannula. This way of administration of a vector into a salivary gland is already known to the skilled person and has already been described (Katano H., et al, (20060, Gene Therapy, 13: 594-601).
  • a vector comprising a nucleic acid molecule encoding the antisense oligonucleotide as identified earlier herein.
  • a preferred vector comprises a nucleic acid molecule encoding an antisense oligonucleotide as identified before, preferably said nucleic acid molecule encoding an antisense oligonucleotide comprises or consists of SEQ ID NO:9 or 20.
  • plasmids, artificial chromosomes, plasmids suitable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein.
  • Preferred for the current invention are those vectors wherein a transcript is in the form of a fusion with the antisense oligonucleotide of the invention with a U7 transcripts, which yield good results for delivering small transcripts (Schumperli, D. Et al, Cell Mol. Life Sci., (2004), 61 :2560- 70). It is within the skill of the artisan to design suitable transcripts. Most preferred is a fusion transcript with a U7 transcript as described in the experimental part.
  • a preferred nucleic acid molecule encoding a mus musculus U7 transcript is represented by a nucleotide sequence comprising or consisting of SEQ ID NO: 10.
  • a preferred nucleic acid molecule encoding a murine U7 transcript is represented by a nucleotide sequence comprising or consisting of SEQ ID NO: 11.
  • the oligonucleotide may also be encoded by the viral vector. Typically this is in the form of an RNA transcript that comprises the sequence of the oligonucleotide in a part of the transcript.
  • a preferred vector comprises a nucleic acid molecule encoding a small nuclear protein U7 linked with a nucleic acid molecule encoding the antisense oligonucleotide of the invention.
  • a preferred nucleic acid molecule encoding a murine small nuclear protein U7 linked with a nucleic acid molecule encoding a murine BAFF antisense oligonucleotide of the invention is represented by a nucleotide sequence comprising or consisting of SEQ ID NO: 12
  • a preferred vector of the invention comprising a nucleic acid molecule encoding a small nuclear protein U7 linked with a nucleic acid molecule encoding the antisense oligonucleotide of the invention is identified as AAV161 or AAV 162 and is represented by a nucleotide sequence identified as SEQ ID NO: 13 or 14. Most preferred is AAV 162.
  • an oligonucleotide When administering an oligonucleotide, it is preferred that an oligonucleotide is dissolved in a solution that is compatible with the delivery method.
  • a solution For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution.
  • an excipient that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell.
  • excipients capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane.
  • Suitable excipients comprise polyethylenimine (PEI), or similar cationic polymers, including
  • PECs polypropyleneimine or polyethylenimine copolymers
  • SAINT- 18 synthetic amphiphils
  • lipofectinTM lipofectinTM
  • DOTAP DOTAP
  • viral capsid proteins that are capable of self assembly into particles that can deliver each constitutent as defined herein to a cell.
  • a preferred oligonucleotide is for preventing or treating the SS in an individual.
  • An individual which may be treated using an oligonucleotide of the invention may already have been diagnosed as having a SS.
  • an individual which may be treated using an oligonucleotide of the invention may not have yet been diagnosed as having a SS but may be an individual having an increased risk of developing a SS in the future given his or her genetic background.
  • a preferred individual is a human being.
  • composition comprising an oligonucleotide or a vector as defined herein.
  • said composition being preferably a pharmaceutical composition said pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.
  • Such a pharmaceutical composition may comprise any pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided.
  • Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000. Each feature of said composition has earlier been defined herein.
  • a vector as defined herein or an antisense oligonucleotide as defined herein wherein the vector or the oligonucleotide is for treating the SS in a patient or for treating a patient predisposed to said syndrome.
  • a method for treating the SS in a patient or for treating a patient predisposed to said syndrome by using the vector as defined herein or an antisense oligonucleotide as defined herein is for alleviating one or more symptom(s) of SS in an individual or alleviate one or more characteristic(s)of a salivary gland cell of said individual, the method comprising administering to said individual an oligonucleotide or a composition or a vector as defined herein.
  • a symptom is dryness for mouth or eyes, fatigue and/or pain that could be assessed by a physician. Dryness for mouth may be assessed using the stimulated saliva test. Dryness for eyes may be assessed using the Schimmer test or tear break-up time. Fatigue and pain may be assessed using a questionnary. Pain may also be assessed using the VAS-score. All these test are known to the skilled person.
  • the diagnosis of SS is preferably assessed based on a global assay as described in Vitali, C. et al (Vitali C, et al (2002), Ann. Rheum. Dis., 61 : 554-8).
  • At least one of these symptoms dryness for mouth, dryness for eyes, fatigue and/or pain and/or the global score as assessed using Vitali C. et al
  • at least one of these symptoms is improved after at least one week, at least one month, 2, 3, 4,5 6, 7, 8, 9, 10, 11, 12 months of treatment by comparison to the same symptom in the same individual at the onset of the treatment.
  • a parameter is the amount of secreted full length BAFF.
  • a method for enhancing, inducing or promoting skipping of an exon from a BAFF mRNA in a cell expressing said mRNA in an individual suffering from SS comprising administering to said individual an oligonucleotide or a composition or a vector as defined herein.
  • an antisense oligonucleotide or a composition or a vector as defined herein is such that in a cell from said individual less secretion of a full length BAFF protein is found compared to the secretion of the same full length BAFF protein in a cell from the same individual at the onset of the treatment.
  • a preferred cell is a human salivary epithelial duct cells from said individual. In this context less is at least 5% less or at least 10%, or at least 15%, or at least 20%>, or at least 30%>, or at least 40%>, or at least 50%, or at least 60% or at least 70%, or at least 80%, or at least 90%, or at least 100% as assessed by Northern blotting. Most preferably, no full length BAFF is detectable outside of the cells. The assessment of a full length BAFF protein may be assessed as earlier defined herein.
  • said method is performed in vivo, for instance using a cell culture or in an animal model such as a NOD mouse or in a human cell from a SS patient or in a salivary gland from a patient or in a patient.
  • a treatment in a use or in a method according to the invention is at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years or more.
  • Each antisense oligonucleotide or vector or composition as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals affected by or at risk of developing SS, and may be administered directly in vivo, ex vivo or in vitro.
  • the frequency of administration of an antisense oligonucleotide or vector or composition of the invention may depend on several parameters such as the age of the patient, the formulation of said molecule.
  • in vitro preferably means a cell free system wherein cellular extracts comprising mRNA are being used and wherein exon skipping may be assessed.
  • in vivo preferably means a cellular model or an animal model or in a human being.
  • Some aspects of the invention concern the use of a vector comprising a nucleic acid molecule as defined above, wherein the vector is a vector that is suitable for gene therapy.
  • Vectors that are suitable for gene therapy are described in Anderson 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60: 249-71; Kay et al, 2001, Nat. Med. 7: 33-40; Russell, 2000, J. Gen. Virol. 81 : 2573-604; Amado and Chen, 1999, Science 285: 674-6; Federico, 1999, Curr. Opin. Biotechnol.10: 448-53; Vigna and Naldini, 2000, J. Gene Med. 2: 308-16; Marin et al, 1997, Mol. Med.
  • a particularly suitable gene therapy vector includes an Adenoviral and Adeno- associated virus (AAV) vector. These vectors infect a wide number of dividing and non-dividing cell types.
  • AAV Adeno-associated virus
  • adenoviral vectors are capable of high levels of transgene expression. However, because of the episomal nature of the adenoviral and AAV vectors after cell entry, these viral vectors are most suited for therapeutic applications requiring only transient expression of the transgene (Russell, 2000, J. Gen. Virol. 81 : 2573-2604; Goncalves, 2005, Virol J. 2(1):43) as indicated above.
  • Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, supra).
  • a preferred retroviral vector for application in the present invention is a lentiviral based expression construct.
  • Lentiviral vectors have the unique ability to infect non- dividing cells (Amado and Chen, 1999 Science 285 : 674-6). Methods for the construction and use of lentiviral based expression constructs are described in U.S. Patent No.'s 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031 and in Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16).
  • gene therapy vectors will be considered expression vectors described above in the sense that they comprise a nucleic acid molecule encoding an antisense oligoncucleotide of the invention to be expressed, whereby said nucleic acid molecule is operably linked to the appropriate regulatory sequences as indicated above.
  • Such regulatory sequence will at least comprise a promoter sequence.
  • Suitable promoters for expression of a nucleotide sequence encoding a polypeptide from gene therapy vectors include e.g.
  • CMV cytomegalovirus
  • LTRs viral long terminal repeat promoters
  • MMLV murine moloney leukaemia virus
  • HTLV-1 hematoma virus
  • SV 40 herpes simplex virus thymidine kinase promoter
  • inducible promoter systems have been described that may be induced by the administration of small organic or inorganic compounds.
  • Such inducible promoters include those controlled by heavy metals, such as the metallothionine promoter (Brinster et al. 1982 Nature 296: 39-42; Mayo et al. 1982 Cell 29: 99-108), RU-486 (a progesterone antagonist) (Wang et al. 1994 Proc. Natl. Acad. Sci. USA 91 : 8180-8184), steroids (Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90: 5603-5607), tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci.
  • tTAER system that is based on the multi- chimeric transactivator composed of a tetR polypeptide, as activation domain of VP16, and a ligand binding domain of an estrogen receptor (Yee et al, 2002, US 6,432,705).
  • a gene therapy vector may optionally comprise a second or one or more nucleic acid molecule coding for a polypeptide.
  • a polypeptide may be a (selectable) marker polypeptide that allows for the identification, selection and/or screening for cells containing the expression construct. Suitable marker proteins for this purpose are e.g.
  • the fluorescent protein GFP and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydro folate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene.
  • HSV thymidine kinase for selection on HAT medium
  • bacterial hygromycin B phosphotransferase for selection on hygromycin B
  • Tn5 aminoglycoside phosphotransferase for selection on G418)
  • DHFR dihydro folate reductase
  • a vector preferably a gene therapy vector is preferably formulated in a pharmaceutical composition as defined herein. Sequence identity
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences (SEQ ID NO), as determined by comparing the full length sequences or part thereof. Preferably the full length of two given SEQ ID NO is being used. Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO, one may replace it by a nucleotide sequence comprising a nucleotide sequence that has at least 60%, 70%, 80%, 90%, 95%, 98%, 99% sequence identity or similarity with it.
  • identity also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215 :403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S . , et al., J. Mol. Biol. 215 :403-410 (1990).
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4.
  • a program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
  • amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.
  • composition or a vector as defined herein may comprise feature(s) than the ones specifically identified, said additional feature(s) not altering the unique characteristic of the invention.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • FIG. 2 Schematic of BAFF and delta BAFF.
  • BAFF and delta BAFF share the same pre-mRNA.
  • Naturally occurring alternative splicing can lead to exon skipping that leads to mRNA encoding for delta BAFF.
  • Figure 3 Exon skipping can be induced by an U7 antisense sequence that covers the exon and results in the removal of 2 introns and 1 exon in the mRNA instead of one intron.
  • Figure 4 Schematic of action of delta BAFF. Delta BAFF binds to BAFF
  • BAFF intracellularly and inhibits secretion of BAFF, therefore BAFF is not available for binding to its receptors.
  • Figure 5 In vitro infection of lymphoma cell line U937 (that constitutively expresses but not secretes BAFF) with a lentiviral construct encoding for a U7 antisense reduced BAFF and increased delta BAFF mRNA over time.
  • Northern blot of U937 cell RNA is shown for day 0, day 5, day 12 and day 14 after infection. Actin was used as a loading control.
  • Figure 6 NOD mice develop lymphocytic foci in the salivary gland with age consisting of B and T cells. Left: focal infiltrates (in purple, arrows) in H&E staining. Middle: focus score with age.
  • each dot represents one mouse.
  • Top right Salivary glands consists of two type of cells; ductal epithelial cells and secretory acinar cells.
  • Figure 7 Schematic of experimental set up. Mice are cannulated at 10 weeks of age with an adeno-associated viral vector (AAV161) or a control vector LacZ and the outcome was analysed at 20 weeks of age.
  • AAV161 adeno-associated viral vector
  • LacZ a control vector LacZ
  • FIG. 8 The presence of BAFF was determined by IHC of paraffin sections. Therapy with the exon skipping inducing adeno-associated vector (from now on named
  • Figure 9 Salivary flow at 20 weeks was increased for the AAV161 treated mice. Mice were anesthesized and salivary flow was induced by injection of pilocarpine. Saliva was collected for 20 minutes and corrected for body weight (BW).
  • Figure 10 Overall inflammation was reduced in AAV161 treated mice. Focus score (average focal infiltrates per mm 2 salivary gland section) was determined at 20 weeks. Treatment with AAV161 reduced focus score.
  • FIG. 11 Sub analysis by IHC of treated and control mice showed dramatic reduction of B220+ cells (left panel) and CD138+ cells in the SG of treated mice. Shown is number of positive cells per mm 2 in submandibular glands of mice).
  • Figure 12 genetic map of AAV161
  • Figure 13 genetic map of AAV 162
  • AAV vectors were generated by packaging AAV2-based recombinant genomes in AAV capsids.
  • the vector used in the study was produced by a three- plasmid transfection protocol. Briefly, HEK293 cells were tri-transfected with the adenovirus helper plasmid pXX6 (Xiao et al, 1998); a pAAV packaging plasmid expressing the rep and cap genes, and the relevant pAAV vector plasmid contaning the U7 antisens construct.
  • the recombinant vector was purified by double-cesium chloride ultracentrifugation followed by dialysis against sterile phosphate-buffered saline (PBS). Viral genomes were quantified by real-time polymerase chain reaction and vector titers are expressed as viral genomes per milliliter (VG/ml).
  • NOD mice Female NOD mice (Jackson Laboratory, Bar Harbor, ME) were kept under specific pathogen free conditions in the animal facilities of the National Institute of Dental and Craniofacial Research (NIDCR). Animal protocols were approved by NIDCR Animal Care and Use Committee and the National Institutes of Health (NIH) Biosafety Committee. Vectors were delivered into the submandibular glands by retrograde instillation.
  • Vector was detected using quantitative-polymerase chain reaction (Q-PCR) on an ABI StepOnePlus Real-Time PCR system (Applied Biosystems, Carlsbad, CA).
  • Primers specific for BAFF and delta-BAFF were used to detect the mRNA of BAFF and delta BAFF specifically.
  • in vitro cells were infected with a lentiviral vector containing the exon skipping inducing U7 sequence and mRNA levels were followed for 2 weeks for levels of specific BAFF and delta BAFF mRNA.
  • RNA was isolated from salivary glands collected in RNAeasy and levels of BAFF and delta BAFF mRNA was measured and compared.
  • Baff6 rev GGCGTAGGTCTTATCAGT (SEQ ID NO:28)
  • Saliva collection was done at 20 weeks of age. Mice were anesthetized as described above and saliva secretion was induced by subcutaneous (sc) injection of pilocarpine (0.5 mg/kg BW; Sigma- Aldrich, St. Louis, MO). Stimulated whole saliva was collected for 20 minutes from the oral cavity with a hematocrit tube (Drummond Scientific Company, Broomall, PA) placed into a preweighed 0.5 ml microcentrifuge tube, and the volume was determined by weight.
  • hematocrit tube Stemond Scientific Company, Broomall, PA
  • One cross sectional part of the submandibular gland was embedded in paraffin and sections were cut at 5 ⁇ .
  • Three sections were stained with hematoxylin and eosin (H&E) and focus score (FS) was determined for each mouse, in which one focus is defined as an average aggregate of 50 or more lymphocytes per 4 mm 2 SG tissue.
  • Other slides were stained with anti-CD 138 (BD, Breda, The Netherlands), anti-BAFF (Alexis Biochemicals, San Diego, CA), and anti- APRIL (Abeam, Cambridge, MA) after heat- induced citrate antigen-retrieval.
  • Another cross-sectional part of the SG was collected and frozen into OCT compound and cut at 5 ⁇ .
  • Salivary gland (SG) lysates made from snap frozen SG in the presence of protease inhibitors were analyzed by western blot and showed reduced BAFF and increased delta BAFF in treated mice compared with control mice.
  • Immunoglobulins (G, A and M) and autoantibodies against Ro- and La-antigens were determined in serum and SG protein extract. SGs were homogenized and the total protein was determined with BCATM protein assay kit (Pierce, Rockford, IL). Samples were analyzed for autoantibodies against SSA/Ro using an in house validated ELISA method to detect 60-kD Ro peptide antibodies.
  • cytokines were determined in homogenized SGs and serum. Cytokines were measured commercially using a multiplex sandwich-ELISA assay (Aushon Biosystem, Billerica, MA). Values were corrected for protein content in the SG protein homogenates. Immunohistochemical stainings
  • cytokines were tested by ELISA and by IHC using specific antibody for each cytokine to confirm local levels.
  • Paraffin-embedded sections were dewaxed in xylene and rehydrated in a gradient of ethanol, followed by incubation with 30% hydrogen peroxidase in 0.1% sodium azide in PBS to block endogenous peroxidase activity.
  • Antigen retrieval was performed by boiling the sections in citrate buffer (pH 6.0) for 10 minutes.
  • TNF, IL-6, IL-10, IL-4, TGFbeta, IL-17, IL-1, IL-12P40 and IFNgamma expression were studied by staining the sections overnight at 4°C with specific anti bodies to the cytokines(Santa Cruz Biotechnology, Santa Cruz, CA and Dako, Glostrup, Denmark) in PBS containing 1 % bovine serum albumin (BSA). Subsequently, the sections were incubated with horseradish peroxidase (HRP)- conjugated secondary antibody in PBS containing 1% BSA and 10% normal human serum, for 30 minutes at room temperature.
  • HRP horseradish peroxidase
  • mice were sacrificed and salivary glands were collected and embedded in paraffin. Sections were stained with a specific antibody for BAFF and analyzed by digital image analysis. In mice treated with the AAV161 vector expression of BAFF was significantly reduced in the SG compared to mice treated with the control vector (P ⁇ 0.04, Figure 8).
  • stimulated saliva flow was measured at 20 weeks of age (10 weeks after delivery of AAV161).
  • BAFF monoclonal antibodies or soluble receptors, even expressed under salivary-specific promoter, may exert a systemic inhibition on membrane-bound BAFF on myeloid and lymphoid cells, and on soluble BAFF expressed by non epithelial cells. Interfering with BAFF mRNA splicing BAFF secretion affects only transduced cells, i.e epithelial cells. Moreover, delta-
  • BAFF is not secreted, its presence at cell surface is discussed, and does not bind to any known receptors. We therefore privileged a different therapeutic approach, using an exon-skipping strategy, to be capable to investigate in vivo the role of BAFF epithelial expression. This selective targeting of epithelial cells was demonstrated by the absence of any impact on serum cytokines, including BAFF, in the present study.
  • the decrease in vitro using myeolomonocytic or lymphoma cells was more pronounced than in vivo. This may be related to the vector used, to a higher number of cells infected in vitro than in vivo, and to the fact that these cells are high producers of BAFF and may be more sensitive to BAFF targeting.
  • the decrease of BAFF could be demonstrated by classical PCR in cell lines but only using qPCR in vivo.
  • the increase of delta-BAFF mRNA level could only be evidenced using classical PCR in cell lines, probably for the same reasons, and due to difficulties to design qPCR specific primers for delta-BAFF (waiting for sequencing results).
  • the second result of the study was the demonstration decreasing BAFF resulted in a marked improvement of dryness.
  • the relatively large amount of mice and the blinded assessment of dryness certainly counterbalance the variability of the dryness in NOD mice.
  • the mechanisms underlying BAFF-decreased related improvement of dryness may be numerous and difficult to dissect given that the pathogenesis of dryness remains uncertain in SS and since the fine analysis of lymphocyte subpopulations and cytokines that can be performed in the periphery is more difficult to achieve in salivary glands. Dryness in SS is envisioned as the result of epithe litis, activation of B-cells, autoantibodies, cytokine storm, matrix disorganization rather than the result of tissular damage due to lymphocytic infiltrates. Of note, B-cell targeting in one controlled trial using rituximab demonstrated a significant effect on dryness.
  • eprastuzumab anti-CD22 antibody
  • a significant decrease of B cells and plasma cells was observed in treated mice.
  • the decrease of plasma cells which is not reported in peripheral blood or spleen after systemic BAFF inhibition, might be related to prevention of the local activation of B cells by epithelial cells (Xu, et al) and formation of germinal- center like structures in salivary glands. It could thus be hypothesized that decreased activation and survival of B lymphocytes, including auto-reactive B cells may improve salivary flow through the decrease of T-cell activation, of B-cell proinflammatory cytokines, such as IL-6.
  • B cells One important pathogenic contribution of B cells to dryness could of course be the secretion of autoantibodies, including anti-M3 muscarinic receptors.
  • APRIL which shares two receptors with BAFF.
  • TACI-Fc dual inhibition of BAFF and APRIL using TACI-Fc resulted in decreased lymphocytic infiltrates but not in improvement of dryness (manuscript submitted). This might be related to a different mechanism of inhibition by a soluble receptor, having less or no impact on the intracellular processing of BAFF and epithelial activation, conversely to the present exon skipping strategy.
  • APRIL may play a rather unexpected protective role in dryness.
  • delta-BAFF might reveal a relevant marker of disease activity in pSS, which lacks such markers; this has to be confirmed in prospective cohorts.
  • BAFF has crucial role on B-cell maturation and activation, and T-cell activation and dependence of BAFF is higher in auto- than allo-reactive B cells. BAFF also play a chemotactic role, which may contribute to the inflammatory infiltrates observed in target organs of autoimmune diseases, including pSS.

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Abstract

L'invention concerne un oligonucléotide antisens apte à induire le saut d'un exon d'un ARNm de BAFF afin de produire une forme tronquée d'une protéine BAFF, lorsqu'il est présent dans une cellule exprimant l'ARNm de BAFF. Cet oligonucléotide peut être utilisé pour le traitement du syndrome de Sjögren.
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WO2018035451A1 (fr) 2016-08-19 2018-02-22 Calimmune, Inc. Méthodes et compositions pour le traitement d'affections à l'aide d'un virus adéno-associé recombinant auto-complémentaire
US11958886B2 (en) 2016-12-07 2024-04-16 University Of Florida Research Foundation, Incorporated IL-1RA cDNAs

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2018035451A1 (fr) 2016-08-19 2018-02-22 Calimmune, Inc. Méthodes et compositions pour le traitement d'affections à l'aide d'un virus adéno-associé recombinant auto-complémentaire
US11207382B2 (en) 2016-08-19 2021-12-28 University Of Florida Research Foundation, Incorporated Compositions for treating conditions using recombinant self-complementary adeno-associated virus
US11958886B2 (en) 2016-12-07 2024-04-16 University Of Florida Research Foundation, Incorporated IL-1RA cDNAs

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