A Helper-Dependent Adenoviral Gene Therapy Delivery and Expression System

Abstract

The present invention relates to gene therapy delivery and expression systems comprising at least one helper-dependent adenoviral vector containing a nucleic acid sequence encoding for proteoglycan 4 (PRG4) or a biologically active fragment thereof. The invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of at least one helper-dependent adenoviral vector containing said nucleic acid sequence encoding for proteoglycan 4 (PRG4), or a homolog thereof from any other species, or a biologically active fragment thereof. The invention also relates to the use of the novel gene therapy delivery and expression system according to the invention for use in the prevention and/or treatment of camptodactyly-arthropathy-coxa vara-pericarditis (CACP), or a musculoskeletal disorder such as a joint disorder or joint disease.

1 . A gene therapy delivery and expression system, comprising at least one helper-dependent adenoviral vector containing a nucleic acid sequence encoding for proteoglycan 4 (PRG4) or a biologically active fragment thereof, left and right adenoviral inverted terminal repeats (LITR and RITR}, adenoviral packaging signal sequences and non-viral, non-coding stuffer nucleic acid sequences. 
     
     
         2 . The gene therapy delivery and expression system according to  claim 1 , wherein PRG4 expression in the at least one helper-dependent adenoviral vector is controlled by a ubiquitous, constitutive promoter selected from the group consisting of elongation factor 1 alpha (EF1 alpha) promoter, cytomegalovirus (CMV) promoter, beta-actin promoter, simian virus 40 (SV40) early promoter, ubiquitin c promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, or other ubiquitous, constitutive promoters. 
     
     
         3 . The gene therapy delivery and expression system according to  claim 1 , wherein the helper-dependent adenoviral vector comprising proteoglycan 4 (PRG4) comprises a nucleic acid sequence set forth in SEQ ID NO 1, or SEQ ID NO 2, or a biologically effective fragment thereof. 
     
     
         4 . The gene therapy delivery and expression system according to  claim 3 , wherein the helper-dependent adenoviral vector comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 2, or a biologically effective fragment thereof. 
     
     
         5 . The gene therapy delivery and expression system according to  claim 1 , wherein the nucleic acid sequence encoding for proteoglycan 4 (PRG4) comprises a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         6 . The gene therapy delivery and expression system according to  claim 5 , wherein the nucleic acid sequence encoding for proteoglycan 4 (PRG4) comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologically effective fragment thereof. 
     
     
         7 . The gene therapy delivery and expression system according to  claim 1 , wherein the amino acid sequence encoding for proteoglycan 4 (PRG4) comprises an amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         8 . The gene therapy delivery and expression system according to  claim 7 , wherein the amino acid sequence encoding for proteoglycan 4 (PRG4) comprises an amino acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with an amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         9 . The gene therapy delivery and expression system according to  claim 1 , wherein the helper-dependent adenoviral vector additionally comprises a nucleic acid sequence encoding for inhibitors of inflammatory and cartilage destructive mediators such as cytokines including Il-1, TNFa, Il-6, Il-7 Il-8, Il-11, Il-15, Il-17, Il-18, Il-21, leukemia inhibitory factor (LIF), oncostatin M; matrix metalloproteases including MMP-1, 3, 9, 13; aggrecanases including ADAMTS-1,4,5; toll-like receptors (TLR) such as TLR2, TLR4; and nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB). 
     
     
         10 . The gene therapy delivery and expression system according to  claim 9 , wherein the helper-dependent adenoviral vector additionally comprises a nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra). 
     
     
         11 . The gene therapy delivery and expression system according to  claim 1 , wherein the delivery and expression system comprises a second helper-dependent adenoviral vector comprising a nucleic acid sequence encoding for inhibitors of inflammatory and cartilage destructive mediators such as cytokines including Il-1, TNFa, Il-6, Il-7 Ik-8, Il-11, Il-15, Il-17, Il-18, Il-21, leukemia inhibitory factor (LIF), oncostatin M; matrix metalloproteases including MMP-1, 3, 9, 13; aggrecanases including ADAMTS-1,4,5; toll-like receptors (TLR) such as TLR2, TLR4; and nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB). 
     
     
         12 . The gene therapy delivery and expression system according to  claim 11 , wherein the delivery and expression system comprises a second helper-dependent adenoviral vector comprising a nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra). 
     
     
         13 . The gene therapy delivery and expression system according to  claim 9 , wherein expression of the inhibitor of inflammatory and cartilage destructive mediators is controlled by an inflammation-inducible promoter selected from the group consisting of NF-κB promoter, interleukin 6(Il-6) promoter, interleukin-1 (Il-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1α (MIP-1α) promoter, or hybrid constructs of the above. 
     
     
         14 . The gene therapy delivery and expression system according to  claim 9 , wherein the helper-dependent adenoviral vector containing the interleukin.-1 receptor antagonist (Il-1Ra) comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7, or SEQ ID NO 8, SEQ ID NO 9, or a biologically effective fragment thereof. 
     
     
         15 . The gene therapy delivery and expression system according to  claim 9 , wherein the nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acid sequence set forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         16 . The gene therapy delivery and expression system according to  claim 9 , wherein the nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 10, or SEQ NO 11, or SEQ ID NO 12, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         17 . The gene therapy delivery and expression system according to  claim 9 , wherein the amino acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra) comprises an amino acid sequence set forth in SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         18 . A pharmaceutical composition, comprising a therapeutically effective amount of at least one helper-dependent adenoviral vector containing a nucleic acid sequence encoding for proteoglycan 4 (PRG4), or a biologically active fragment thereof. 
     
     
         19 . The pharmaceutical composition according to  claim 18 , wherein PRG4 expression in the at least one helper-dependent adenoviral vector is controlled by a ubiquitous, constitutive promoter selected from the group consisting of elongation factor alpha (EF1 alpha) promoter, cytomegalovirus (CMV) promoter, beta-actin promoter, simian virus 40 (SV40) early promoter, ubiquitin c promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, or other ubiquitous, constitutive promoters. 
     
     
         20 . The pharmaceutical composition according to  claim 18 , wherein the helper-dependent adenoviral vector comprising proteoglycan 4 (PRG4) comprises a nucleic acid sequence set forth in SEQ ID NO 1, or SEQ ID NO 2, or a biologically effective fragment thereof. 
     
     
         21 . The pharmaceutical composition according to  claim 20 , wherein the helper-dependent adenoviral vector comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 2, or a biologically effective fragment thereof. 
     
     
         22 . The pharmaceutical composition according to  claim 18 , wherein the nucleic acid sequence encoding for proteoglycan 4 (PRG4) comprises a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         23 . The pharmaceutical composition according to  claim 22 , wherein the nucleic acid sequence encoding for proteoglycan (PRG4) comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologically effective fragment thereof. 
     
     
         24 . The pharmaceutical composition according to  claim 18 , wherein the amino acid sequence encoding for proteoglycan 4 (PRG4) comprises an amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         25 . The pharmaceutical composition according to  claim 24 , wherein the amino acid sequence encoding for proteoglycan 4 (PRG4) comprises an amino acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with an amino acid sequence set forth in SEQ ID NO 5, or SEQ ID NO 6, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         26 . The pharmaceutical composition according to  claim 18 , wherein the helper-dependent adenoviral vector additionally comprises a nucleic acid sequence encoding for inhibitors of inflammatory and cartilage destructive mediators such as cytokines including Il-1, TNFα, Il-6, Il-7, Il-8, Il-11, Il-15, Il-17, Il-18, Il-21, leukemia inhibitory factor (LIF), oncostatin M; matrix metalloproteases including MMP-1, 3, 9, 13; aggrecanases including ADAMTS-1,4, 5; toll-like receptors (TLR) such as TLR2, TLR4; and nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB). 
     
     
         27 . The pharmaceutical composition according to  claim 26 , wherein the helper-dependent adenoviral vector additionally comprises a nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra). 
     
     
         28 . The pharmaceutical composition according to  claim 18 , wherein the delivery and expression system comprises a second helper-dependent adenoviral vector comprising a nucleic acid sequence encoding encoding for inhibitors of inflammatory and cartilage destructive mediators such as cytokines including Il-1, TNFa, Il-6, Il-7, Il-8, Il-11, Il-15, Il-17, Il-18, Il-21, leukemia inhibitory factor (LIF), oncostatin M; matrix metalloproteases including MMP-1, 3, 9, 13; aggrecanases including ADAMTS-1,4, 5; toll-like receptors (TLR) such as TLR2, TLR4; and nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB). 
     
     
         29 . The pharmaceutical composition according to  claim 28 , wherein the delivery and expression system comprises a second helper-dependent adenoviral vector comprising a nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra). 
     
     
         30 . The pharmaceutical composition according to  claim 26 , wherein expression of the inhibitor of inflammatory and cartilage destructive mediators is controlled by an inflammation-inducible promoter selected from the group consisting of NF-κB promoter, interleukin 6 (II-6) promoter, interleukin-1 (Il-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1α (MIP-1α) promoter, or hybrid constructs of the above. 
     
     
         31 . The pharmaceutical composition according to  claim 26 , wherein the helper-dependent adenoviral vector containing the interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7, or SEQ ID NO 8, SEQ ID NO 9, or a biologically effective fragment thereof. 
     
     
         32 . The pharmaceutical composition according to  claim 26 , wherein the nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acid sequence set forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         33 . The pharmaceutical composition according to  claim 26 , wherein the nucleic acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra) comprises a nucleic acid sequence which has at least 50%, 60%, 70%, 80% or 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         34 . The pharmaceutical composition according to  claim 26 , wherein the amino acid sequence encoding for interleukin-1 receptor antagonist (Il-1Ra) comprises an amino acid sequence set forth in SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 or a biologically active fragment thereof, or a homolog thereof from any other species. 
     
     
         35 . A gene therapy delivery and expression system according to  claim 1 , for use in the prevention and/or treatment of camptodactyly-arthropathy-coxa vera-pericarditis (CACP) syndrome. 
     
     
         36 . A gene therapy delivery and expression system according to  claim 1 , for use in the prevention and/or treatment of a musculoskeletal disorder. 
     
     
         37 . The gene therapy delivery and expression system according to  claim 1 , for use in the prevention and/or treatment of a joint disorder or disease. 
     
     
         38 . The gene therapy delivery and expression system according to  claim 36 , wherein the disorder is selected from the group consisting of arthropathies, all types of arthritis, including arthritis-related disorders, osteoarthritis, rheumatoid arthritis, gout and pseudo-gout, septic arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, Still's disease, Reiter's syndrome, or tendinopathies including tendonitis, tendinosis, tenosynovitis; synovial disorders including synovitis; Bursa disorders including bursitis; equine musculoskeletal disorders including bone spavin, navicular syndrome, osselet. 
     
     
         39 . The gene therapy delivery and expression system according to  claim 37 , wherein the disease or disorder is selected from the group consisting of arthropathies, all types of arthritis, including arthritis-related disorders, osteoarthritis, rheumatoid arthritis, gout and pseudo-gout, septic arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, Still's disease, Reiter's syndrome, or tendinopathies including tendonitis, tendinosis, tenosynovitis; synovial disorders including synovitis; Bursa disorders including bursitis; equine musculoskeletal disorders including bone spavin, navicular syndrome, osselet.