Helper-dependent adenoviral gene therapy delivery and expression system
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 pharmaceutical composition, comprising a therapeutically effective amount of at least one helper-dependent adenoviral vector containing a nucleic acid sequence encoding proteoglycan 4 (PRG4), or a biologically active fragment thereof, which has chondoprotective activity, a left and a right adenoviral inverted terminal repeats (L ITR and R ITR), adenoviral packaging signal sequences and non-viral, non-coding stuffer nucleic acid sequences,
wherein
i. the at least one helper-dependent adenoviral vector additionally comprises a nucleic acid sequence encoding interleukin-1 receptor antagonist (II-1Ra), or
ii the composition comprises a second helper-dependent adenoviral vector comprising a nucleic acid sequence encoding Il-1Ra;
wherein PRG4 expression is controlled by an elongation factor 1 alpha (EF 1 alpha) promoter and expression of Il-1Ra is controlled by an NF-κB promoter, and
wherein the at least one helper-dependent adenoviral vector containing a nucleic acid sequence encoding PRG4, comprises a nucleic acid sequence which has at least 90% sequence homology with the nucleic acid sequence set forth in SEQ ID NO: 1, or SEQ ID NO: 2.
2. The pharmaceutical composition according to claim 1 , wherein the nucleic acid sequence encoding 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, or a nucleic acid sequence which has at least 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologically active fragment thereof, wherein the PRG4 encoded by the biologically active fragment of SEQ ID NO 3, or SEQ ID NO 4, or a nucleic acid sequence which has at least 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 3, or SEQ ID NO 4, or a biologically active fragment thereof, has chondoprotective activity.
3. The pharmaceutical composition according to claim 1 , wherein the amino acid sequence of 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, or an amino acid sequence which has at least 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, wherein the amino acid sequence of PRG4, which comprises a biologically active fragment of the amino acid SEQ ID NO 5, or SEQ ID NO 6, or has at least 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, has chondoprotective activity.
4. The pharmaceutical composition according to claim 1 , wherein the at least one helper-dependent adenoviral vector comprising the nucleic acid sequence encoding Il-1Ra, comprises a nucleic acid sequence which has at least 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or a biologically active fragment thereof, wherein the at least one helper-dependent adenoviral vector comprising the nucleic acid sequence encoding II-1Ra, which comprises a nucleic acid sequence which has at least 90% sequence homology with a nucleic acid sequence set forth in SEQ ID NO 7, or SEQ ID NO 8, or SEQ ID NO 9, or a biologically active fragment thereof, has II-1Ra activity of inhibiting inflammatory and cartilage destructive mediators.
5. The pharmaceutical composition according to claim 1 , wherein the nucleic acid sequence encoding 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 wherein the nucleic acid sequence encoding for Il-1Ra comprises a nucleic acid sequence which has at least 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, wherein the Il-1Ra encoded by the biologically active fragment of SEQ ID NO 10, or SEQ ID NO 11, or SEQ ID NO 12, or the nucleic acid sequence which has at least 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 the biologically active fragment thereof, mediates II1-Ra activity of inhibiting inflammatory and cartilage destructive mediators.
6. The pharmaceutical composition according to claim 1 , wherein the amino acid sequence of Il-1Ra comprises the amino acid sequence of human IL-1Ra (SEQ ID NO 13), murine IL-1Ra (SEQ ID NO: 14), equine IL-1Ra (SEQ ID NO: 15), or a biologically active fragment thereof, or a homolog thereof from any other species, which has Il-1Ra activity of inhibiting inflammatory and cartilage destructive mediators.