Patent Application
20240294924
The present invention relates to antisense-oligonucleotides capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, and salts and optical isomers of said antisense-oligonucleotides for prevention of kidney dysfunction promoted by endothelial dysfunction.
End stage renal disease (ESRD) is a worldwide public health problem with an enormous financial burden for healthcare systems. Endothelial dysfunction is known to induce microangiopathy in the kidney often coupled with podocyte foot process effacement, which is the key process of nephropathy. In the developed and developing world, diabetes is the major cause of microangiopathy, resulting in ESRD. Hyperglycemia in diabetes leads to many complications such as hypertension and nephropathy. Under the condition, hypertension is a major pathogenic factor driving the development of microvascular dysfunction in diabetes. In the kidney, increased blood pressure attenuates glomerular filtration barriers between the blood and urinary space, resulting in proteinuria, the major risk of developing ESRD. Compromised kidney filtration function in diabetic nephropathy is the major cause of kidney failure.
Glomeruli are specialized filtration barriers between the blood and urinary space, comprised of podocytes, glomerular basement membrane (GBM) and fenestrated glomerular endothelial cells (GECs). The morphology of healthy podocyte foot processes is essential for kidney filtration function. Effacement of foot processes is observed in most proteinuric diseases including diabetic nephropathy.
Recent studies have shown that nephrin, a transmembrane protein localized at slit diaphragm of foot process, and nephrin phosphorylation are important for maintenance of the podocyte foot processes. Decreased expression of nephrin and nephrin phosphorylation are known to result in renal dysfunction characterized by pathologic foot process remodeling. Loss of nephrin results in podocyte effacement and causes proteinuria, which is often seen in the onset of ESRD. In diabetes, hyperglycemia causes endothelial dysfunction, leading to foot process effacement, suggesting cellular communication from endothelial cells (ECs) to podocytes is important for development of the disease. However, the molecular link connecting endothelial cells to podocytes across the glomerular basement membrane (GBM) is not known.
Current treatment options for diabetic nephropathy are limited to control of blood pressure and blood glucose levels. It is believed that hypertension in diabetes causes microvascular dysfunction, including the glomerulus, leading to proteinuria. However, the effect of those treatments is limited.
The international patent application WO2004/080418A2 discloses nucleic acid compounds, e.g. antisense-oligonucleotides (ASOs) for inhibiting EphrinB2 or EphB4 expression for treating cancer or angiogenesis-associated diseases. The international patent application WO2007/038395A2 discloses nucleic acid compounds, e.g. antisense-oligonucleotides (ASOs) for inhibiting EphrinB2 or EphB4 expression for treating viral infections. In order to investigate the inhibitory effect of ASOs on EphrinB2 expression, the nucleotide sequence of the protein-coding region of the EphrinB2 transcript was subdivided into sections of 20 consecutive nucleotides and 51 antisense-oligonucleotides (ASOs) consisting of 20 nucleotides targeting each of said sections of the protein-coding region of the EphrinB2 transcript were tested. Of the 51 antisense-oligonucleotides (ASOs), 8 ASOs targeting a section within the 1st to 262nd nucleotide of the protein coding region have resulted in the strongest inhibitory effect.
The US patent application US2004/110150A1 is directed to compounds, compositions and methods for modulating the expression of Ephrin B2 and discloses antisense-oligonucleotides consisting of 20 nucleotides in length composed of a central “gap” region consisting of ten 2′-deoxynucleotides which is flanked on both sides by five-nucleotide wings that are composed of 2′-methoxyethyl nucleotides. The US patent application US2004/110150A1 discloses antisense-oligonucleotides targeting different regions of genomic sequence and different regions of the Ephrin B2 transcript such as regions corresponding to the 3′-untranslated region (UTR) or protein coding region of the Ephrin B2 transcript, but also other regions such as intron regions or intron:exon junctions, start codon or stop codon. Of the antisense-oligonucleotides (ASOs), ASOs targeting a section within the 1st to 262nd nucleotide of the protein coding region, a section within the 510th to 900th, within the 1475th to 1490th, within the 1755th to 1775th, within the 1937th to 1957th and within the 2420th to 2440th nucleotide of the 3′-UTR have resulted in the strongest inhibitory effect.
It is the objective of the present invention to provide pharmaceutically active agents, especially for use in the treatment of nephropathy coupled with podocyte foot process effacement, such as diabetic nephropathy as well as compositions comprising at least one of those compounds as pharmaceutically active ingredients. The objective of the present application is also to provide pharmaceutically active compounds for use in controlling nephrin function in podocytes.
The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.
The present invention relates to antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
It is an essential aspect of the present invention that the target sequence for the antisense-oligonucleotides described herein for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy is located within a region of the gene encoding Efnb2 or a region of the mRNA encoding Efnb2 comprising a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Such target sequences are particularly advantageous because they exhibit the important cross-reactivity between the two species. The antisense oligonucleotides of the present invention consist of 10 to 28 nucleotides. A sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse has the great advantage that ASOs with a maximum length of 28 nucleotides can hybridize with a 100% conserved sequence over the full length.
Antisense-oligonucleotide comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse have been found to be particularly advantageous for inhibiting ephrin-B2 expression in endothelial cells. It has been surprisingly found that antisense-oligonucleotides of the present invention are therapeutically effective for restoring nephrin expression and phosphorylation and for restoring podocyte foot process effacement. Surprisingly, the effect of the antisense-oligonucleotides of the present invention is far stronger than that of the current first line drugs for use in the treatment of nephropathy coupled with podocyte foot process effacement.
Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 is within a protein-coding region of the gene encoding Efnb2, or the mRNA encoding Efnb2. Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 is within an open reading frame of the gene encoding Efnb2, or the mRNA encoding Efnb2. Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 is within a 3′-untranslated region (UTR) of the mRNA encoding Efnb2.
Antisense-oligonucleotides containing LNAs (LNA®: Locked Nucleic Acids) have been found to be particularly important to provide the desired inhibitory effect on ephrinB2 expression when the antisense-oligonucleotides target the sequences AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3) that are located within a region of a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Thus, according to the present invention at least two of the 10 to 28 nucleotides are LNAs. It is further preferred that at least four of the 10 to 28 nucleotides are LNAs. In preferred embodiments the antisense-oligonucleotide has a gapmer structure with 1 to 5 LNA units at the 3′ terminal end and 1 to 5 LNA units at the 5′ terminal end.
In preferred embodiments the antisense-oligonucleotide hybridizes selectively only with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. In further preferred embodiments the antisense-oligonucleotide hybridizes selectively only with the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. In further preferred embodiments the antisense-oligonucleotide hybridizes selectively only with the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) of the region of the gene encoding Efnb2 or of the region of the mRNA encoding the Efnb2.
In preferred embodiments, the antisense oligonucleotides of the present invention bind with 100% complementarity to the regions of the gene encoding Efnb2 or to the mRNA encoding Efnb2 as described herein and do not bind to any other region in the human transcriptome.
In preferred embodiments, the antisense-oligonucleotide consists of a sequence of 10 to 16 nucleotides of the sequence AGGTTAGGGCTGAATT (Seq. ID No. 4) which is complementary to the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. Preferably, the antisense-oligonucleotide consists of a sequence of 10 to 16 nucleotides of the sequence AGGTTAGGGCTGAATT selected from:
In preferred embodiments, the antisense-oligonucleotide consists of a sequence of 10 to 16 nucleotides of the sequence TACAAGCAAGGCATTT (Seq. ID No. 5) which is complementary to the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. Preferably, the antisense-oligonucleotide consists of a sequence of 10 to 16 nucleotides of the sequence TACAAGCAAGGCATTT selected from:
In preferred embodiments, the antisense-oligonucleotide consists of a sequence of 10 to 16 nucleotides of the sequence ACATTGCAAAATTCAG (Seq. ID No. 6) which is complementary to the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. Preferably, the antisense-oligonucleotide consists of a sequence of 10 to 16 nucleotides of the sequence ACATTGCAAAATTCAG selected from:
The antisense-oligonucleotides of the present invention are particularly useful as pharmaceutical active agents for suppression of ephrin-B2 function or secretion providing renal protective effects. Thus, antisense-oligonucleotides of the present invention are suitable for use in controlling nephrin function, preferably for use in the prophylaxis and treatment of proteinuria in diabetes and/or of diabetic nephropathy.
The present invention further relates to a pharmaceutical composition containing at least one antisense-oligonucleotide together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, solvent or diluent.
The present invention further relates to a method of treating an animal (or human or patient) having a disease selected from nephropathy and/or diabetic proteinuria and/or diabetic nephropathy comprising administering to said animal a therapeutically or prophylactically effective amount of at least one antisense-oligonucleotide of the present invention or pharmaceutical composition according to the present invention.
The present invention further relates to a method of inhibiting the expression of Ephrin-B2 in cells or tissues comprising incubating said cells or tissues with an effective amount of at least one antisense oligonucleotide according to the present invention.
The present invention further relates to a method of restoring nephrin function in cells or tissues comprising incubating said cells or tissues with an effective amount of at least one antisense-oligonucleotide of the present invention.
Surprisingly, it has been found that suppression of ephrin-B2 in endothelial cells strongly restore podocyte foot process effacement and kidney function induced by decreased nephrin expression.
EphB receptor tyrosine kinases and their transmembrane ligand, ephrin-B2 mediate cell-to-cell contact dependent signaling and thus regulate cell migration and cytoskeletal organization in many different cell types and tissues. The binding of ephrin-B2 induces clustering and auto-phosphorylation of EphB receptors, resulting in downstream signal activation in the cells expressing EphBs (termed as “forward signaling”). Simultaneously, the cytoplasmic tail of ephrin-B2, via its C-terminal PDZ binding motif or phosphorylation of tyrosine residues, engages in “reverse signaling”. In the vasculature, ephrin-B2 controls VEGF receptor trafficking and downstream signaling, thereby regulating endothelial sprouting behavior during angiogenesis. In mature tissues, ephrin-B2 is a marker of arterial and arterial derived ECs, including glomerular endothelial cells (GECs). However, the role of ephrin-B2 in mature endothelial cells (ECs) is largely unknown.
The inventors have surprisingly found that cell-to-cell contact independent ephrin-B2/EphB4 forward signaling mediated by extracellular vesicles such as exosomes plays a pivotal role in cellular communication from endothelial cells (ECs) to podocytes.
It has been found that this signaling pathway is over-activated in the diabetic condition. This pathway is over-activated in diabetes to cause podocyte foot process effacement, and suppression of ephrin-B2/EphB4 by cell type specific gene deletion has been found to prevent glomerular dysfunction in mice. Proteinuria and podocyte effacement in diabetic mice are restored in both ephrin-B2 endothelial specific and EphB4 podocyte specific inducible knockout mice.
Surprisingly, it has been found that antisense-oligonucleotides capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, and salts and optical isomers of said antisense-oligonucleotides solve the above objective.
Thus, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides.
Preferably, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides.
More preferably, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
Slightly reworded, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with an exon region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
Antisense-oligonucleotide comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse have been found to be particularly advantageous for inhibiting ephrin-B2 expression in endothelial cells. It has been surprisingly found that antisense-oligonucleotides of the present invention are therapeutically effective for restoring nephrin expression and phosphorylation and for restoring podocyte foot process effacement. Surprisingly, the effect of the antisense-oligonucleotides of the present invention is far stronger than that of the current first line drugs for use in the treatment of nephropathy coupled with podocyte foot process effacement.
Thus, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, and wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells.
Thus, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, and wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes.
Thus, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, and wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes.
Thus, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, and wherein said antisense-oligonucleotide restores podocyte foot process effacement.
More preferably, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells, and wherein said antisense-oligonucleotide restores podocyte foot process effacement.
More preferably, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells, and wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes
More preferably, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein said antisense-oligonucleotide restores podocyte foot process effacement, and wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes
Most preferably, the present invention preferably relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells, wherein said antisense-oligonucleotide restores podocyte foot process effacement, and wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes
Preferably, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence, AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides; and wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferably, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells. Preferably, wherein said antisense-oligonucleotide restores podocyte foot process effacement. Preferably, wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes.
More preferably, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence, AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides; and wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells, wherein said antisense-oligonucleotide restores podocyte foot process effacement, and wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation.
Preferably, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence, AAATGCCTTGCTTGTA (Seq. ID No. 2), or CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotides; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 is within a 3′-untranslated region (UTR) of the mRNA encoding Efnb2.
The present invention relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide.
Preferably, the present invention relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
Preferably, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells. Preferably, wherein said antisense-oligonucleotide restores podocyte foot process effacement. Preferably, wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes.
Preferably, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotides; and wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
With other words, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a region of the open reading frame of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the open reading frame of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotides. Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
Slightly reworded, the present invention relates to antisense-oligonucleotides consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides nucleotides are LNAs, wherein the antisense-oligonucleotides are capable of hybridizing with a protein coding region of the gene encoding Efnb2, or with a protein coding region of the mRNA encoding Efnb2, wherein the region of the protein coding region of the gene encoding Efnb2, or the protein coding region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotides. Preferably, the protein coding region of the gene encoding Efnb2, or the protein coding region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 is within a protein coding region of the gene encoding Efnb2, or a protein coding region of the mRNA.
Preferably, an antisense-oligonucleotide as described herein hybridizes selectively only with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. Preferably, the antisense-oligonucleotide oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, hybridizes selectively only with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2.
The complementary sequence to the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) is AGGTTAGGGCTGAATT (5′-3′) (Seq. ID No. 4). The sequence AATTCAGCCCTAACCT (Seq. ID No. 1) is also written in 5′-3′ direction. Preferred antisense-oligonucleotides of the present invention comprise a sequence that overlaps with or corresponds to the sequence AGGTTAGGGCTGAATT (5′-3′) (Seq. ID No. 4). The antisense-oligonucleotides of the present invention consist of 10 to 28 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence AGGTTAGGGCTGAATT (5′-3′) (Seq. ID No. 4). Thus, an antisense-oligonucleotide comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) is particularly preferred. Thus, the antisense-oligonucleotides according to the invention hybridize with at least a sequence of at least 10 consecutive nucleotides within the sequence AATTCAGCCCTAACCT (Seq. ID No. 1). In preferred embodiments, at least said 10 consecutive nucleotides of the antisense-oligonucleotide are complementary, preferably 100% complementary, to a region within the target sequence AATTCAGCCCTAACCT (Seq. ID No. 1).
Thus, the present invention therefore preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein at least the sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) are complementary, preferably 100% complementary, to a region within the sequence AATTCAGCCCTAACCT (Seq. ID No. 1).
The present invention further relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), and the antisense-oligonucleotides comprise a sequence capable of hybridizing with said sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), and salts and optical isomers of said antisense-oligonucleotide.
Preferably, the present invention therefore relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferably, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells. Preferably, wherein said antisense-oligonucleotide restores podocyte foot process effacement. Preferably, wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes.
Preferably, the antisense-oligonucleotide hybridizes selectively only with the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2. Preferably, the antisense-oligonucleotide oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, hybridizes selectively only with the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) of the region of the gene encoding Efnb2, or of the region of the mRNA encoding the Efnb2.
The complementary sequence to the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) is TACAAGCAAGGCATTT (5′-3′) (Seq. ID No. 5). The sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) is also written in 5′-3′ direction. Preferred antisense-oligonucleotides of the present invention comprise a sequence that overlaps with or corresponds to the sequence TACAAGCAAGGCATTT (5′-3′) (Seq. ID No. 4). According to the present invention the antisense-oligonucleotides consist of 10 to 28 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence TACAAGCAAGGCATTT (5′-3′) (Seq. ID No. 5). Thus, an antisense-oligonucleotide comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) is particularly preferred. Thus, the antisense-oligonucleotides according to the invention hybridize with at least a sequence of at least 10 consecutive nucleotides within target sequence AAATGCCTTGCTTGTA (Seq. ID No. 2). In preferred embodiments, at least said 10 consecutive nucleotides are complementary, preferably 100% complementary, to a region within the target sequence AAATGCCTTGCTTGTA (Seq. ID No. 2).
Thus, the present invention therefore preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AAATGCCTTGCTTGTA (Seq. ID No. 2), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein at least the sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) are complementary, preferably 100% complementary, to a region within the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2).
The present invention therefore relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotide.
Preferably, the present invention therefore relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferably, wherein said antisense-oligonucleotide inhibits ephrin-B2 expression in endothelial cells. Preferably, wherein said antisense-oligonucleotide restores podocyte foot process effacement. Preferably, wherein said antisense-oligonucleotide restores nephrin expression. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation. Preferably, wherein said antisense-oligonucleotide restores nephrin expression and phosphorylation in podocytes. Preferably, wherein said antisense-oligonucleotide increases nephrin expression. Preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation. More preferably, wherein said antisense-oligonucleotide increases nephrin expression and phosphorylation in podocytes.
Preferably, the antisense-oligonucleotide hybridizes selectively only with the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) of the region of the gene encoding Efnb2 or of the region of the mRNA encoding the Efnb2. Preferably, the antisense-oligonucleotide oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, hybridizes selectively only with the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) of the region of the gene encoding Efnb2 or of the region of the mRNA encoding the Efnb2.
The complementary sequence to the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) is ACATTGCAAAATTCAG (5′-3′) (Seq. ID No. 6). The sequence CTGAATTTTGCAATGT (Seq. ID No. 3) is also written in 5′-3′ direction. Preferred antisense-oligonucleotides of the present invention comprise a sequence that overlaps or corresponds to the sequence ACATTGCAAAATTCAG (5′-3′) (Seq. ID No. 6). According to the present invention the antisense-oligonucleotides consist of 10 to 28 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence ACATTGCAAAATTCAG (5′-3′) (Seq. ID No. 6). Thus, an antisense-oligonucleotide comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) is particularly preferred. Thus, the antisense-oligonucleotides according to the invention hybridize with at least a sequence of at least 10 consecutive nucleotides within target sequence CTGAATTTTGCAATGT (Seq. ID No. 3). In preferred embodiments, at least said 10 consecutive nucleotides are complementary, preferably 100% complementary, to a region within the target sequence CTGAATTTTGCAATGT (Seq. ID No. 3).
Thus, the present invention therefore preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence CTGAATTTTGCAATGT (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, wherein at least the sequence of at least 10 consecutive nucleotides capable of hybridizing with the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) are complementary, preferably 100% complementary, to a region within the sequence CTGAATTTTGCAATGT (Seq. ID No. 3).
While the role of ephrin-B2 as a regulator of VEGF receptor signaling has been established during angiogenesis, the role of ephrin-B2 in mature endothelial cells (ECs) is largely unknown. Previous work has shown that VEGF signaling in GECs regulates endothelial fenestration, a critical characteristic of GEC.
To gain further insight into the role of endothelial ephrin-B2 in mature GECs, the inventors examined the expression of ephrin-B2 in the glomerulus. The signal corresponding to the immunoreactivity of an ephrin-B2 antibody in the glomerulus was observed not only in CD31, a marker of endothelial cells (ECs), positive structures but also in the nephrin positive podocytes (
Since ephrin-B2 expression was reduced in the podocytes in the EC specific KO mice, the inventors have examined the phenotype of podocytes in the Efnb2iΔEC mice. Surprisingly, significantly increased nephrin expression was observed in the Efnb2iΔEC mice compared to control mice using immunostaining and western blotting analysis (
Podocytes and ECs in the glomerulus are known to be physically segregated by the glomerular basement membrane (GBM). To examine whether ephrin-B2 in ECs is able to activate EphB4 on podocytes without cell-to-cell contact formation, the inventors first ectopically expressed N-terminal GFP-flag-tagged ephrin-B2 in cultured cells (
To confirm ephrin-B2 secretion in vivo, the inventors analyzed mice expressing CFP-ephrin-B2 in ECs. Overexpression of ephrin-B2 in endothelial cells causes toxicity in mice. In VE-cadherin-tTA/tetO—CFP-Efnb2 mice, the administration of tetracycline to pregnant female mice circumvents the embryonic lethality of ephrin-B2 overexpression. After birth, mosaic expression of CFP-ephrin-B2 in ECs was induced by withdrawal of tetracycline treatment. Only mice displaying low levels of ephrin-B2 overexpression reached adulthood. Staining with an anti-GFP antibody (recognizing CFP) revealed the mosaic expression of CFP in CD31 positive ECs. Although the low levels of ephrin-B2 overexpression might not be enough to affect nephrin expression in these mice, a specific signal corresponding to CFP-ephrin-B2 was also observed in nephrin positive podocytes (
To investigate the link between ephrin-B2/EphB4 forward signaling and nephrin, the inventors next carried out immunoprecipitation of EphB4 from kidney tissue and confirmed nephrin/EphB4 complex formation (
In agreement with an inhibitory role of ephrin-B2 on nephrin phosphorylation, mRNA of ephrin-B2 is increased in patients suffering from type 2 diabetic nephropathy compared to those suffering from type 2 diabetes. Additionally, EphB4 is shown to be a marker for advanced type 1 diabetic state presenting retinopathy and nephropathy.
To gain further insight into the role of ephrin-B2 on diabetic nephropathy, the inventors examined immunostaining of ephrin-B2 in renal biopsy in diabetic patients. While three patients were diagnosed with hyperoxaluria, other three were diagnosed with diabetic nephropathy (DN). In the DN patient tissues, signal against ephrin-B2 co-localized with nephrin in those glomeruli was stronger than that in biopsy from the hyperoxaluria patients (
The inventors next examined if suppressing ephrin-B2/EphB4 forward signaling in mice with diabetic nephropathy would restore nephrin function and podocyte foot process architecture and would be beneficial for preventing diabetic kidney failure.
To address this, the inventors employed diabetic mice models with both the ephrin-B2 or EphB4 mutants. To examine the effect of ephrin-B2 loss of function in diabetes, control and Efnb2iΔEC mice on the C57BL/6 background were injected with streptozotocin at 5 weeks of age and fed with high fat diet. C57BL/6 control and Efnb2iΔEC mice developed a mild diabetic phenotype after 18 weeks. HE staining of the kidneys, blood glucose level, and body weight between control and Efnb2iΔEC diabetic mice were not different. Serum creatinine levels were decreased in Efnb2iΔEC mice although not at significant levels (
Taken together, these results indicate that the suppression of ephrin-B2/EphB4 forward signaling across the GBM has renal protective effects by controlling nephrin function in the podocytes.
The inventors examined the effect of antisense oligonucleotides (ASOs) on ephrin-B2 expression. Different sequences against ephrin-B2 (Efnb2ASOs) were examined with the cultured ECs. Among them, treatment of ECs with 4 different ASOs resulted in effective knockdown of ephrin-B2 (
The inventors of the present invention have examined and intensively analysed the genomic sequence of the gene encoding Efnb2 and the sequence of the mRNA encoding Efnb2 to determine the regions providing the most effective target sequence for antisense-oligonucleotides for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy. It has been found that therapeutically effective antisense-oligonucleotides for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy relate to antisense-oligonucleotides that are capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the mRNA encoding Efnb2 comprises a sequence that is 100% conserved between human and mouse. The antisense-oligonucleotides according to the invention consist of 10 to 28 nucleotides. A sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse has the advantage that ASOs with a lengths of 28 nucleotides can hybridize with a 100% conserved sequence over the full length. Thus, a region of the gene encoding Efnb2, or a region of the mRNA encoding Efnb2 is strongly preferred, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. With other words, a region of the gene encoding Efnb2, or a region of the mRNA encoding Efnb2 is strongly preferred, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between Homo sapiens and Mus musculus.
Thus, an essential aspect of the present invention is that the target sequence for antisense-oligonucleotides for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy is located within a region of the gene encoding Efnb2 or a region of the mRNA encoding Efnb2 comprising a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 36 consecutive nucleotides that is 100% conserved between human and mouse. Preferably, the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 46 consecutive nucleotides that is 100% conserved between human and mouse. Said target sequences have been found to be particularly advantageous because they exhibit the important cross-reactivity between the two species.
In order to identify potential target sequences of at least 28 consecutive nucleotides that are 100% conserved between human and mouse, the genomic sequence of Homo sapiens as set forth in Seq. ID No. 101 (representing the Homo sapiens chromosome 13, GRCh38.p13 Primary Assembly) has been aligned with the genomic sequence of Mus musculus as set forth in Seq. ID No. 289 (representing Mus musculus strain C57BL/6J chromosome 8, GRCm38.p6 C57BL/6J). The inventors have found that preferred regions within the gene encoding Efnb2, or the mRNA encoding Efnb2 for antisense-oligonucleotides for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy relate to antisense-oligonucleotides that are capable of hybridizing with an exon region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2. The inventors of the present invention have further investigated the genomic sequence of the gene encoding Efnb2 and the sequence of the mRNA encoding Efnb2 to determine the regions having the greatest potential to provide advantageous target sequences for antisense-oligonucleotides for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy and have found that antisense-oligonucleotides are most promising that are capable of hybridizing with an open reading frame (ORF) of the gene encoding Efnb2, or with a protein coding region of the mRNA encoding Efnb2 or with a region of the gene encoding Efnb2 or the region of the mRNA encoding Efnb2 within the 3′-untranslated region (UTR) of the mRNA encoding Efnb2.
The exons of protein-coding genes contain the open reading frame (ORF) and additionally the 5′ and 3′ untranslated region (UTR) from the terminal exons. In human/Homo sapiens the mRNA transcript of ephrin B2 has in total 5 exons. The first exon (Seq. ID No. 90) encodes the 5′-untranslated region (UTR) and a part of the protein-coding sequence, the second (Seq. ID No. 91), third (Seq. ID No. 92) and fourth (Seq. ID No. 93) exons encode a part of the protein-coding sequence and the fifth exon (Seq. ID No. 94) encodes a part of the protein-coding sequence and the 3′-untranslated region (UTR) (Seq. ID No. 296). Different transcripts of human the mRNA are known in the art. The Sequence of Seq. ID No. 98 represents Homo sapiens ephrin B2 (EFNB2), transcript variant 1, mRNA written in the DNA code. The sequence of Seq. ID No. 99 represents Homo sapiens ephrin B2 (EFNB2), transcript variant 2, mRNA written in the DNA code. The Seq. ID No. 100 represents Homo sapiens ephrin B2 (EFNB2), transcript variant 3, mRNA written in the DNA code. The inventors have identified that the mRNA transcript variant 2 lacks the sequence of the second exon region and the mRNA transcript variant 3 lacks the sequence of the third exon region.
In the next step, the inventors have identified sequences that are 100% conserved between humans and mice within an exon region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2. The nucleotide sequence of the coding region (Seq. ID No. 295) of mRNA, Homo sapiens ephrin B2 (EFNB2), transcript variant 1 has been aligned with the sequence of the coding region of the mRNA, Mus musculus ephrin B2 (EFNB2). The inventors have identified sequences of at least 28 consecutive nucleotides that are 100% conserved between human and mouse within the coding region of the EphrinB2 transcript. It has been found that the sequence of the second exon (Seq. ID No. 91) and the third exon (Seq. ID No. 92) include preferred sequences of at least 28, more preferably at least 36, more preferably at least 46 nucleotides that are 100% conserved between humans and mice. However, only the second exon region is contained in all three mRNA transcript variants 1, 2 and 3 of EphrinB2 in Homo sapiens. Thus, the second exon region has been found to be a particularly preferred target for antisense-oligonucleotides for use in the prophylaxis and treatment of nephropathy and/or proteinuria in diabetes and/or diabetic nephropathy.
The inventors examined the effect of antisense oligonucleotides (ASOs) on ephrin-B2 expression by examining different sequences against the sequence of the second exon region of the gene encoding ephrin-B2 (Efnb2ASOs) with cultured ECs. Thereby, the inventors have found that the inventive ASO of the sequence AGGTTAGGGCTGAATT (5′-3′) (Seq. ID No. 4) comprising 3 LNAs at the 3′-terminal end and 3 LNAs at the 5′-terminal end resulted in effective knockdown of ephrin-B2 (
The sequence AATTCAGCCCTAACCT (Seq. ID No. 1) is advantageously 100% conserved between humans and mice within a region of at least 28 nucleotides. Moreover, the sequence AATTCAGCCCTAACCT (Seq. ID No. 1) is not only 100% conserved between humans and mice within a region of at least 28 nucleotides, but is also 100% conserved between, for example, human and rat, human and chimpanzee, human and macaque within a region of at least 28 nucleotides.
Preferably, the present invention therefore relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human, mouse, rat, chimpanzee and macaque.
Thus, the present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within a second exon region (Seq. ID No. 291) of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse.
The prior art WO2004/080418A2 and WO2007/038395A2 disclose 51 antisense-oligonucleotides (ASOs) consisting of 20 nucleotides targeting the protein-coding region of the EphrinB2 transcript. Of the 51 antisense-oligonucleotides (ASOs), 8 ASOs targeting a section within the 1st to 262nd nucleotide of the protein coding sequence have resulted in the strongest inhibitory effect. Thereby, the section between the 131st to 262nd is within the second exon region of the gene encoding ephrin-B2 (Efnb2ASOs). Of the 8 ASOs targeting a section within the 1st to 262nd nucleotide of the protein coding sequence, 4 ASOs targeting the second exon region of the gene encoding ephrin-B2 (Efnb2ASOs) have resulted in the strongest inhibitory effect. The target sequence of the present invention AATTCAGCCCTAACCT (Seq. ID No. 1) is located between the 356th and 372nd nucleotide of the protein coding sequence of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2. Antisense-oligonucleotides (ASOs) of WO2004/080418A2 and WO2007/038395A2 targeting a region between the 356th and 415th nucleotide of the protein coding sequence have shown only medium inhibitory effect in this area. Also US2004/110150A1 discloses ASOs targeting a section within the 1st to 262nd nucleotide of the protein coding sequence resulting in the strongest inhibitory effect on EphrinB2 expression. US2004/110150A1 also discloses that (ASOs) within the section from the 263rd to 415th nucleotide of the protein coding sequence have resulted in medium inhibitory effect on EphrinB2 expression.
However, the antisense-oligonucleotide of the present invention consists of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs. It has been found that antisense-oligonucleotides containing LNAs (LNA®: Locked Nucleic Acids) are particularly important to provide the desired inhibitory effect on ephrinB2 expression when the antisense-oligonucleotides of the present invention target the sequences AATTCAGCCCTAACCT (Seq. ID No. 1) that is located within a region of a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, and wherein the region of the gene encoding Efnb2 is within a second exon region (Seq. ID No. 291) of the gene encoding Efnb2. Thus, contrary to the teachings of the prior art, the antisense oligonucleotides of the present invention enable effective inhibition of ephrinB2 expression by targeting the sequence AATTCAGCCCTAACCT (Seq. ID No. 1).
The inventors have further examined the effect of antisense oligonucleotides (ASOs) on ephrin-B2 expression by examining different sequences against the sequence of the fifth exon region of the gene encoding ephrin-B2 (Efnb2ASOs) or in particular the 3′-unstranslated region (UTR) with cultured ECs. Thereby, the inventors have found that the ASO of the sequence TACAAGCAAGGCATTT (5′-3′) (Seq. ID No. 5) and ACATTGCAAAATTCAG 5′-3′) (Seq. ID No. 6) both comprising three LNAs on the 3′-terminal end and three LNAs on the 5′-terminal end resulted in effective knockdown of ephrin-B2 (
Thus, the inventors of the present invention have identified the two sequences AAATGCCTTGCTTGTA (Seq. ID No. 2) and CTGAATTTTGCAATGT (Seq. ID No. 3) as the most advantageous target sequence within the 3′-untranslated region (UTR) which is located within the fifth exon region of the gene encoding Efnb2, or within a region of the mRNA encoding Efnb2.
The sequences AAATGCCTTGCTTGTA (Seq. ID No. 2) and CTGAATTTTGCAATGT (Seq. ID No. 3) are advantageously 100% conserved between human and mice within a region of at least 28 nucleotides. Moreover, the sequences AAATGCCTTGCTTGTA (Seq. ID No. 2) and CTGAATTTTGCAATGT (Seq. ID No. 3) are not only 100% conserved between humans and mice within a region of at least 28 nucleotides but are also 100% conserved between, for example, human and chimpanzee within a region of at least 28 nucleotides.
Thus, the present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding the Efnb2, comprises the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) or ACATTGCAAAATTCAG (Seq. ID No. 3), and the antisense-oligonucleotides comprise a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) or ACATTGCAAAATTCAG (Seq. ID No. 3), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within a 3′-unstranslated region (UTR) (Seq. ID No. 296) of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human, mouse and chimpanzee.
The prior art US2004/110150A1 discloses antisense-oligonucleotides targeting different regions of genomic sequence and different regions of the Ephrin B2 mRNA transcript such as regions corresponding to the 3′-untranslated region (UTR) or protein coding region of the Ephrin B2 mRNA transcript. Of the antisense-oligonucleotides (ASOs), ASOs targeting a section within the 510th to 900th, within the 1475th to 1490th, within the 1755th to 1775th, within the 1937th to 1957th and within the 2420th to 2440th nucleotide of the 3′-UTR have resulted in the strongest inhibitory effect on EphrinB2 expression. However, any of the antisense-oligonucleotides (ASOs) targeting the 3′-UTR that have shown strong inhibitory effect target a sequence of the 3′-UTR that is 100% conserved between human and mouse. Thus, the ASOs of US2004/110150A1 targeting a sequence within the 3′-UTR of the mRNA encoding Efnb2 are not suitable for the present invention. US2004/110150A1 completely fails to disclose ASOs that can hybridize with the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) or ACATTGCAAAATTCAG (Seq. ID No. 3).
The antisense-oligonucleotide of the present invention consists of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs. It has been found that antisense-oligonucleotides containing LNAs (LNA®: Locked Nucleic Acids) are particularly important to provide the desired inhibitory effect on ephrinB2 expression, when the antisense-oligonucleotides of the present invention target the sequences AAATGCCTTGCTTGTA (Seq. ID No. 2) or ACATTGCAAAATTCAG (Seq. ID No. 3). that are located within a region of a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse, and wherein the region of the gene encoding Efnb2 is within a 3′-untranslated region (UTR) (Seq. ID No. 296) of the mRNA encoding Efnb2. Thus, contrary to the teachings of the prior art, the antisense oligonucleotides of the present invention enable effective inhibition of ephrinB2 expression by targeting the sequence AAATGCCTTGCTTGTA (Seq. ID No. 2) or ACATTGCAAAATTCAG (Seq. ID No. 3).
Preferably, the antisense-oligonucleotide of the present invention consists of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs. Thus, the antisense-oligonucleotides of the present invention preferably comprise 2 to 10 LNA units, more preferably 3 to 9 LNA units and still more preferably 4 to 8 LNA units and also preferably at least 6 non-LNA units, more preferably at least 7 non-LNA units and most preferably at least 8 non-LNA units. The non-LNA units are preferably DNA units. The LNA units are preferably positioned at the 3′ terminal end (also named 3′ terminus) and the 5′ terminal end (also named 5′ terminus). Preferably at least one and more preferably at least two LNA units are present at the 3′ terminal end and/or at the 5′ terminal end.
Thus, preferred are antisense-oligonucleotides which contain 3 to 10 LNA units and which especially contain 1 to 5 LNA units at the 5′ terminal end and 1 to 5 LNA units at the 3′ terminal end of the antisense-oligonucleotide and between the LNA units at least 7 and more preferably at least 8 DNA units. Thus, in preferred embodiments the antisense-oligonucleotides have a gapmer structure with 1 to 5 LNA units at the 3′ terminal end and 1 to 5 LNA units at the 5′ terminal end.
Moreover, the antisense-oligonucleotides may contain common nucleobases such as adenine, guanine, cytosine, thymine and uracil as well as common derivatives thereof. The antisense-oligonucleotides of the present invention may also contain modified internucleotide bridges such as phosphorothioate or phosphorodithioate instead of phosphate bridges. Such modifications may be present only in the LNA segments or only in the non-LNA segment of the antisense-oligonucleotide or both.
Preferred are the following antisense-oligonucleotides (Table 1) consisting of 10 to 16 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence AGGTTAGGGCTGAATT (Seq. ID No. 4):
More preferred are the following antisense-oligonucleotides (Table 2) consisting of 10 to 16 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence AGGTTAGGGCTGAATT (Seq. ID No. 4):
Preferred are antisense-oligonucleotides of general formula (S1) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S1) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable, but not limited to said LNA nucleotides (LNA units), and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable, but are not limited to said “Internucleotide Linkages (IL)”.
More preferably the antisense-oligonucleotide of general formula (S1) has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus. Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are gapmers of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Further preferred are antisense-oligonucleotides of the formula (S1):
wherein
Further preferred are antisense-oligonucleotides of the formula (S1):
wherein
In preferred embodiments N1 represents GGT- and N2 represents -A. In another preferred embodiments N1 represents GT- and N2 represents -AA. In another preferred embodiments N1 represents T- and N2 represents -AAT.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S1):
wherein
Preferred are the flowing antisense-oligonucleotides (Table 3):
Preferred are antisense-oligonucleotides of general formula (S1A) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S1A) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable, but not limited to said LNA nucleotides (LNA units), and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable, but are not limited to said “Internucleotide Linkages (IL)”.
More preferably the antisense-oligonucleotide of general formula (S1A) has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus. Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are gapmers of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Further preferred are antisense-oligonucleotides of the formula (S1A):
wherein
Further preferred are antisense-oligonucleotides of the formula (S1A):
wherein
In preferred embodiments N1A represents AGG- and N2A represents -G. In another preferred embodiments N1A represents GG- and N2A represents -GA. In another preferred embodiments N1A represents G- and N2A represents -GAA.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S1A):
wherein N1A represents: GG- and N2A represents: -GA.
Preferred are the following antisense-oligonucleotides Table 4):
Preferred are antisense-oligonucleotides of general formula (S1B) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S1B) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable, but not limited to said LNA nucleotides (LNA units), and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable, but are not limited to said “Internucleotide Linkages (IL)”.
More preferably the antisense-oligonucleotide of general formula (S1B) has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus. Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are gapmers of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Further preferred are antisense-oligonucleotides of the formula (S1B):
wherein
Further preferred are antisense-oligonucleotides of the formula (S1B):
wherein
In preferred embodiments N1B represents GAG- and N2B represents -T. In another preferred embodiments N1B represents AG- and N2B represents -TG. In another preferred embodiments N1B represents G- and N2B represents -TGA.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S1B):
wherein N1B represents G- and N2B represents -TGA.
Preferred following antisense-oligonucleotides (Table 5):
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N1-TAGGGCTG-N3-3′ (Seq. ID No. 7) or 5′-N1A-TTAGGGCT-N2A-3′ (Seq. ID No. 34) or 5′-N1B-GTTAGGGC-N2B-3′ (Seq. ID No. 35), wherein
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N1-TAGGGCTG-N2-3′ (Seq. ID No. 7) or 5′-N1A-TTAGGGCT-N2A-3′ (Seq. ID No. 34) or 5′-N1B-GTTAGGGC-N2B-3′ (Seq. ID No. 35), wherein the residues N1, N2, N1A, N2A, N1B and N2B have the meanings especially the further limited meanings as disclosed herein and salts and optical isomers of said antisense-oligonucleotide.
Surprisingly, it has been found that antisense-oligonucleotides consisting of 10 to 16 nucleotides, preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides, wherein at least two of the 10 to 16 nucleotides, preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides, are LNAs, have the advantage that any reagent for transfection like lipofectamine has to be used in the experiments and “free uptake” is possible.
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCT (Seq. ID No. 9), GTTAGGGCTG (Seq. ID No. 10), TTAGGGCTGA (Seq. ID No. 11), AGGTTAGGGCTG (Seq. ID No. 15), GGTTAGGGCTGA (Seq. ID No. 16), GTTAGGGCTGAA (Seq. ID No. 17), TTAGGGCTGAAT (Seq. ID No. 18), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), GGTTAGGGCTG (Seq. ID No. 21), GTTAGGGCTGA (Seq. ID No. 22), TTAGGGCTGAA (Seq. ID No. 23), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), GTTAGGGCTGAAT (Seq. ID No. 28), TTAGGGCTGAATT (Seq. ID No. 29), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), GTTAGGGCTGAATT (Seq. ID No. 32), AGGTTAGGGCTGAATT (Seq. ID No. 4) and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCT (Seq. ID No. 9), GTTAGGGCTG (Seq. ID No. 10), TTAGGGCTGA (Seq. ID No. 11), GGTTAGGGCTGA (Seq. ID No. 16), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), GGTTAGGGCTG (Seq. ID No. 21), GTTAGGGCTGA (Seq. ID No. 22), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), AGGTTAGGGCTGAATT (Seq. ID No. 4) and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence AGGTTAGGGCTG (Seq. ID No. 15), GGTTAGGGCTGA (Seq. ID No. 16), GTTAGGGCTGAA (Seq. ID No. 17), TTAGGGCTGAAT (Seq. ID No. 18), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), GTTAGGGCTGAAT (Seq. ID No. 28), TTAGGGCTGAATT (Seq. ID No. 29), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), GTTAGGGCTGAATT (Seq. ID No. 32), AGGTTAGGGCTGAATT (Seq. ID No. 4) and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCTGA (Seq. ID No. 16), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), AGGTTAGGGCTGAATT (Seq. ID No. 4) and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence AGGTTAGGGCTG (Seq. ID No. 15), GGTTAGGGCTGA (Seq. ID No. 16), GTTAGGGCTGAA (Seq. ID No. 17), TTAGGGCTGAAT (Seq. ID No. 18), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), GTTAGGGCTGAAT (Seq. ID No. 28), TTAGGGCTGAATT (Seq. ID No. 29), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), GTTAGGGCTGAATT (Seq. ID No. 32), and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCTGA (Seq. ID No. 16), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), and salts and optical isomers of said antisense-oligonucleotide.
The antisense-oligonucleotides of formula S1, S1A or S1B in form of gapmers (LNA segment 1—DNA segment—LNA segment 2) contain an LNA segment at the 5′ terminal end consisting of 2 to 5, preferably 2 to 4 LNA units and contain an LNA segment at the 3′ terminal end consisting of 2 to 5, preferably 2 to 4 LNA units and between the two LNA segments one DNA segment consisting of 6 to 14, preferably 7 to 12 and more preferably 8 to 11 DNA units.
The antisense-oligonucleotides of formula S1, S1A or S1B contain the LNA nucleotides (LNA units) as disclosed herein, especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed in the chapter “Preferred LNAs”. The LNA units and the DNA units may comprise standard nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), but may also contain modified nucleobases as disclosed in the chapter “Nucleobases”. The antisense-oligonucleotides of formula S1, S1A or S1B or the LNA segments and the DNA segment of the antisense-oligonucleotide may contain any internucleotide linkage as disclosed herein and especially these disclosed in the chapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotides of formula S1, S1A or S1B may optionally also contain endgroups at the 3′ terminal end and/or the 5′ terminal end and especially these disclosed in the chapter “Terminal groups”.
Experiments have shown that modified nucleobases do not considerably increase or change the activity of the inventive antisense-oligonucleotides in regard to tested neurological and oncological indications. The modified nucleobases 5-methylcytosine or 2-aminoadenine have been demonstrated to further increase the activity of the antisense-oligonucleotides of formula S1, S1A or S1B especially if 5-methylcytosine is used in the LNA nucleotides only or in the LNA nucleotides and in the DNA nucleotides and/or if 2-aminoadenine is used in the DNA nucleotides and not in the LNA nucleotides.
As LNA units for the antisense-oligonucleotides of formula S1, S1A or S1B especially β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-ENA (b5), β-D-(NH)-LNA (b6), β-D-(NCH3)-LNA (b7), β-D-(ONH)-LNA (b8) and β-D-(ONCH3)-LNA (b9) are preferred. Experiments have been shown that all of these LNA units b1, b2, b4, b5, b6, b7, b8, and b9 can be synthesized with the required effort and lead to antisense-oligonucleotides of comparable stability and activity. However based on the experiments the LNA units b1, b2, b4, b5, b6, and b7 are further preferred. Still further preferred are the LNA units b1, b2, b4, b6, and b7, and even more preferred are the LNA units b1 and b4 and most preferred also in regard to the complexity of the chemical synthesis is the β-D-oxy-LNA (b1).
So far no special 3′ terminal group or 5′ terminal group could be found which remarkably had changed or increased the stability or activity for oncological or neurological indications, so that 3′ and 5′ end groups are possible but not explicitly preferred.
Various internucleotide bridges or internucleotide linkages are possible. In the formulae disclosed herein the internucleotide linkage IL is represented by -IL′-Y—.
Thus, IL=-IL′-Y—=—X″—P(═X′)(X−)—Y—, wherein IL is preferably selected form the group consisting of:
Preferred are the internucleotide linkages IL selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —S—P(O)(O−)—O—, —S—P(O)(S−)—O—, —O—P(O)(O−)—S—, —O—P(O)(S−)—S—, —S—P(O)(O−)—S—, —O—P(O)(OCH3)—O—, —O—P(O)(NH(CH3))—O—, —O—P(O)[N(CH3)2]—O—, —O—P(O)(OCH2CH2OCH3)—O—, and more preferred selected from —O—P(O)(O)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —S—P(O)(O−)—O—, —S—P(O)(S−)—O—, —O—P(O)(O−)—S—, —O—P(O)(S−)—S—, —S—P(O)(O−)—S—, and still more preferred selected from —O—P(O)(O)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, and most preferably selected from —O—P(O)(O−)—O—and —O—P(O)(S−)—O—.
Thus, the present invention preferably relates to an antisense-oligonucleotide in form of a gapmer consisting of 10 to 28 nucleotides, preferably 10 to 18 nucleotides, more preferably 10 to 16, and still more preferably 12 to 16 or 12 to 14 nucleotides and 1 to 5 of these nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the 3′ terminal end of the antisense-oligonucleotide are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the 3′ terminal end a sequence of at least 6, preferably 7 and more preferably 8 DNA nucleotides is present, and the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N1-TAGGGCTG-N2-3′ (Seq. ID No. 7) or 5′-N1A-TTAGGGCT-N2A-3′ (Seq. ID No. 34) or 5′-N1B-GTTAGGGC-N2B-3′ (Seq. ID No. 35), wherein the residues N1, N2, N1A, N2A, N1B and N2B have the meanings especially the further limited meanings as disclosed herein, and
Still further preferred, the present invention relates to an antisense-oligonucleotide in form of a gapmer consisting of 10 to 28 nucleotides, preferably 10 to 18 nucleotides, more preferably 10 to 16, and still more preferably 12 to 16 or 12 to 14 nucleotides and 1 to 5 of these nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the 3′ terminal end of the antisense-oligonucleotide are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the 3′ terminal end a sequence of at least 6, preferably 7 and more preferably 8 DNA nucleotides is present, and the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N1-TAGGGCTG-N2-3′ (Seq. ID No. 7) or 5′-N1A-TTAGGGCT-N2A-3′ (Seq. ID No. 34) or 5′-N1B-GTTAGGGC-N2B-3′ (Seq. ID No. 35), wherein the residues N1, N2, N1A, N2A, N1B and N2B have the meanings especially the further limited meanings as disclosed herein, and and the LNA nucleotides are selected from β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-(NH)-LNA (b6), and β-D-(NCH3)-LNA (b7); and
Especially preferred are the gapmer antisense-oligonucleotides of Table 1 or Table 2 or Tables 3-5 containing a segment of 2 to 5, preferably 2 to 4 and more preferably 2 to 3 LNA units at the 3′ terminus and a segment of 2 to 5, preferably 2 to 4 and more preferably 2 to 3 LNA units at the 5′ terminus and a segment of at least 6, preferably 7 and more preferably 8 DNA units between the two segments of LNA units, wherein the LNA units are selected from β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-(NH)-LNA (b6), and β-D-(NCH3)-LNA (b7) and the internucleotide linkages are selected from phosphate, phosphorothioate and phosphorodithioate. Such preferred antisense-oligonucleotides may not contain any modified 3′ and 5′ terminal end or may not contain any 3′ and 5′ terminal group and may as modified nucleobase contain 5-methylcytosine in the LNA units, preferably all the LNA units and/or 2-aminoadenine in some or all DNA units and/or 5-methylcytosine in some or all DNA units.
Preferred are the following antisense-oligonucleotides (Table 6) consisting of 10 to 16 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence TACAAGCAAGGCATTT (Seq. ID No. 5):
More preferred are the following antisense-oligonucleotides (Table 7) consisting of 10 to 16 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence TACAAGCAAGGCATTT (Seq. D No. 5):
Preferred are also antisense-oligonucleotides of general formula (S2) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S2) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable.
More preferably the antisense-oligonucleotide has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus.
Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are GapmeRs of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Thus, preferred are also antisense-oligonucleotides of general formula (S2)
wherein
Further preferred are antisense-oligonucleotides of the formula (S2):
wherein
In preferred embodiments N3 represents ACA- and N4 represents -A. In another preferred embodiments N3 represents CA- and N4 represents -AT. In another preferred embodiments N3 represents A- and N4 represents -ATT.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S2):
wherein
Preferred are the following antisense-oligonucleotides (Table 8):
Preferred are also antisense-oligonucleotides of general formula (S2A) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S2A) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable.
More preferably the antisense-oligonucleotide has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus.
Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are GapmeRs of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Thus, preferred are also antisense-oligonucleotides of general formula (S2A):
wherein
Further preferred are also antisense-oligonucleotides of general formula (S2A):
wherein
In preferred embodiments N3A represents TAC- and N4A represents -C. In another preferred embodiments N3A represents AC- and N4A represents -CA. In another preferred embodiments N3A represents C- and N4A represents -CAT.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S2A):
wherein
Preferred are the following antisense-oligonucleotides (Table 9):
Preferred are also antisense-oligonucleotides of general formula (S2B) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S2B) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable.
More preferably the antisense-oligonucleotide has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus.
Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are GapmeRs of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Thus, preferred are also antisense-oligonucleotides of general formula (S2B):
wherein
Further preferred are also antisense-oligonucleotides of general formula (S2B):
wherein
In preferred embodiments N3B represents CAA- and N4B represents -T. In another preferred embodiments N3B represents AA- and N4B represents -TT. In another preferred embodiments N3B represents A- and N4B represents -T.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S2B):
wherein
Preferred are the following antisense-oligonucleotides (Table 10):
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N3-AGCAAGGC-N4-3′(Seq. ID No. 64) or 5′-N3A-AAGCAAGG-N4A-3′ (Seq. ID No. 65) or GCAAGGCA-NW3 (Seq. ID No. 66), wherein
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N3-AGCAAGGC-N4-3′ (Seq. ID No. 64) or 5′-N3A-AAGCAAGG-N4A-3′ (Seq. ID No. 65) or 5′-N3B-GCAAGGCA-N4B-3′ (Seq. ID No. 66), wherein the residues N3, N4, N3A, N4A, N3B and N4B have the meanings especially the further limited meanings as disclosed herein and salts and optical isomers of said antisense-oligonucleotide.
Surprisingly, it has been found that antisense-oligonucleotides consisting of 10 to 16 nucleotides, preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides, wherein at least two of the 10 to 16 nucleotides, preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides, are LNAs, have the advantage that any reagent for transfection like lipofectamine has to be used and “free uptake” is possible.
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence CAAGCAAGGC (Seq. ID No. 39), AAGCAAGGCA (Seq. ID No. 40), AGCAAGGCAT (Seq. ID No. 41), TACAAGCAAGGC (Seq. ID No. 44), ACAAGCAAGGCA (Seq. ID No. 45), CAAGCAAGGCAT (Seq. ID No. 46), AAGCAAGGCATT (Seq. ID No. 47), AGCAAGGCATTT (Seq. ID No. 48), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), ACAAGCAAGGC (Seq. ID No. 52), CAAGCAAGGCA (Seq. ID No. 53), AAGCAAGGCAT (Seq. ID No. 54), AGCAAGGCATT (Seq. ID No. 55), TACAAGCAAGGCA (Seq. ID No. 57), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), AAGCAAGGCATTT (Seq. ID No. 60), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62) CAAGCAAGGCATTT (Seq. ID No. 63) TACAAGCAAGGCATTT (Seq. ID No. 5) and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence CAAGCAAGGC (Seq. ID No. 39), AAGCAAGGCA (Seq. ID No. 40), AGCAAGGCAT (Seq. ID No. 41), CAAGCAAGGCAT (Seq. ID No. 46), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), CAAGCAAGGCA (Seq. ID No. 53), AAGCAAGGCAT (Seq. ID No. 54), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62) CAAGCAAGGCATTT (Seq. ID No. 63) TACAAGCAAGGCATTT (Seq. ID No. 5) and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TACAAGCAAGGC (Seq. ID No. 44), ACAAGCAAGGCA (Seq. ID No. 45), CAAGCAAGGCAT (Seq. ID No. 46), AAGCAAGGCATT (Seq. ID No. 47), AGCAAGGCATTT (Seq. ID No. 48), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), TACAAGCAAGGCA (Seq. ID No. 57), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), AAGCAAGGCATTT (Seq. ID No. 60), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63), TACAAGCAAGGCATTT (Seq. ID No. 5) and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence CAAGCAAGGCAT (Seq. ID No. 46), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63), TACAAGCAAGGCATTT (Seq. ID No. 5) and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TACAAGCAAGGC (Seq. ID No. 44), ACAAGCAAGGCA (Seq. ID No. 45), CAAGCAAGGCAT (Seq. ID No. 46), AAGCAAGGCATT (Seq. ID No. 47), AGCAAGGCATTT (Seq. ID No. 48), TACAAGCAAGGCA (Seq. ID No. 57), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), AAGCAAGGCATTT (Seq. ID No. 60), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63) and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence CAAGCAAGGCAT (Seq. ID No. 46), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63) and salts and optical isomers of said antisense-oligonucleotide.
The antisense-oligonucleotides of formula S2, S2A or S2B in form of gapmers (LNA segment 1—DNA segment—LNA segment 2) contain an LNA segment at the 5′ terminal end consisting of 2 to 5, preferably 2 to 4 LNA units and contain an LNA segment at the 3′ terminal end consisting of 2 to 5, preferably 2 to 4 LNA units and between the two LNA segments one DNA segment consisting of 6 to 14, preferably 7 to 12 and more preferably 8 to 11 DNA units.
The antisense-oligonucleotides of formula S2, S2A or S2B contain the LNA nucleotides (LNA units) as disclosed herein, especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed in the chapter “Preferred LNAs”. The LNA units and the DNA units may comprise standard nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), but may also contain modified nucleobases as disclosed in the chapter “Nucleobases”. The antisense-oligonucleotides of formula S2, S2A or S2B or the LNA segments and the DNA segment of the antisense-oligonucleotide may contain any internucleotide linkage as disclosed herein and especially these disclosed in the chapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotides of formula S2, S2A or S2B may optionally also contain endgroups at the 3′ terminal end and/or the 5′ terminal end and especially these disclosed in the chapter “Terminal groups”.
Experiments have shown that modified nucleobases do not considerably increase or change the activity of the inventive antisense-oligonucleotides in regard to tested neurological and oncological indications. The modified nucleobases 5-methylcytosine or 2-aminoadenine have been demonstrated to further increase the activity of the antisense-oligonucleotides of formula S2, S2A or S2B especially if 5-methylcytosine is used in the LNA nucleotides only or in the LNA nucleotides and in the DNA nucleotides and/or if 2-aminoadenine is used in the DNA nucleotides and not in the LNA nucleotides.
As LNA units for the antisense-oligonucleotides of formula S2, S2A or S2B especially β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-ENA (b5), β-D-(NH)-LNA (b6), β-D-(NCH3)-LNA (b7), β-D-(ONH)-LNA (b8) and β-D-(ONCH3)-LNA (b9) are preferred. Experiments have been shown that all of these LNA units b1, b2, b4, b5, b6, b7, b8, and b9 can be synthesized with the required effort and lead to antisense-oligonucleotides of comparable stability and activity. However based on the experiments the LNA units b1, b2, b4, b5, b6, and b7 are further preferred. Still further preferred are the LNA units b1, b2, b4, b6, and b7, and even more preferred are the LNA units b1 and b4 and most preferred also in regard to the complexity of the chemical synthesis is the β-D-oxy-LNA (b1).
So far no special 3′ terminal group or 5′ terminal group could be found which remarkably had changed or increased the stability or activity for oncological or neurological indications, so that 3′ and 5′ end groups are possible but not explicitly preferred.
Various internucleotide bridges or internucleotide linkages are possible. In the formulae disclosed herein the internucleotide linkage IL is represented by -IL′-Y—. Thus, IL=-IL′-Y—=—X″—P(═X′)(X−)—Y—, wherein IL is preferably selected form the group consisting of:
Preferred are the internucleotide linkages IL selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —S—P(O)(O−)—O—, —S—P(O)(S−)—O—, —O—P(O)(O−)—S—, —O—P(O)(S−)—S—, —S—P(O)(O−)—S—, —O—P(O)(OCH3)—O—, —O—P(O)(NH(CH3))—O—, —O—P(O)[N(CH3)2]—O—, —O—P(O)(OCH2CH2OCH3)—O—, and more preferred selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —S—P(O)(O−)—O—, —S—P(O)(S−)—O—, —O—P(O)(O−)—S—, —O—P(O)(S−)—S—, —S—P(O)(O−)—S—, and still more preferred selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, and most preferably selected from —O—P(O)(O−)—O—and —O—P(O)(S−)—O—.
Thus, the present invention preferably relates to an antisense-oligonucleotide in form of a gapmer consisting of 10 to 28 nucleotides, preferably 10 to 18 nucleotides, more preferably 10 to 16, and still more preferably 12 to 16 or 12 to 14 nucleotides and 1 to 5 of these nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the 3′ terminal end of the antisense-oligonucleotide are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the 3′ terminal end a sequence of at least 6, preferably 7 and more preferably 8 DNA nucleotides is present, and the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N3-AGCAAGGC-N4-3′ (Seq. ID No. 64) or 5′-N3A-AAGCAAGG-N4A-3′ (Seq. ID No. 65) or 5′-N5B-NGCAAGGCA-N4B-3′ (Seq. ID No. 66), wherein the residues N3, N4, N3A, N4A, N3B and N4B have the meanings especially the further limited meanings as disclosed herein, and
Still further preferred, the present invention relates to an antisense-oligonucleotide in form of a gapmer consisting of 10 to 28 nucleotides, preferably 10 to 18 nucleotides, more preferably 10 to 16, and still more preferably 12 to 16 or 12 to 14 nucleotides and 1 to 5 of these nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the 3′ terminal end of the antisense-oligonucleotide are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the 3′ terminal end a sequence of at least 6, preferably 7 and more preferably 8 DNA nucleotides is present, and the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N3-AGCAAGGC-N4-3′ (Seq. ID No. 64) or 5′-N3A-AAGCAAGG-N4A-3′ (Seq. ID No. 65) or 5′-N3B-GCAAGGCA-N4B-3′ (Seq. ID No. 66), wherein the residues N3, N4, N3A, N4A, N3B and N4B have the meanings especially the further limited meanings as disclosed herein, and the LNA nucleotides are selected from β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-(NH)-LNA (b6), and β-D-(NCH3)-LNA (b7); and
Especially preferred are the gapmer antisense-oligonucleotides of Table 6 or Table 7 or Tables 8-10 containing a segment of 2 to 5, preferably 2 to 4 and more preferably 2 to 3 LNA units at the 3′ terminus and a segment of 2 to 5, preferably 2 to 4 and more preferably 2 to 3 LNA units at the 5′ terminus and a segment of at least 6, preferably 7 and more preferably 8 DNA units between the two segments of LNA units, wherein the LNA units are selected from β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-(NH)-LNA (b6), and β-D-(NCH3)-LNA (b7) and the internucleotide linkages are selected from phosphate, phosphorothioate and phosphorodithioate. Such preferred antisense-oligonucleotides may not contain any modified 3′ and 5′ terminal end or may not contain any 3′ and 5′ terminal group and may as modified nucleobase contain 5-methylcytosine in the LNA units, preferably all the LNA units and/or 2-aminoadenine in some or all DNA units and/or 5-methylcytosine in some or all DNA units.
Preferred are the following antisense-oligonucleotides (Table 11) consisting of 10 to 16 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence ACATTGCAAAATTCAG (Seq. ID No. 6):
More preferred are the following antisense-oligonucleotides (Table 12) consisting of 10 to 16 nucleotides and preferably comprise a sequence of at least 10 consecutive nucleotides that corresponds to a sequence of 10 consecutive nucleotides of the sequence ACATTGCAAAATTCAG (Seq. ID No. 6):
Preferred are also antisense-oligonucleotides of the general formula (S3) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S3) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable.
More preferably the antisense-oligonucleotide has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus.
Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 and still more preferable 12 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are GapmeRs of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Thus, preferred are also antisense-oligonucleotides of general formula (S3)
wherein
Further preferred are antisense-oligonucleotides of general formula (S3)
wherein
In preferred embodiments N5 represents ATT- and N6 represents -C. In another preferred embodiments N5 represents TT- and N6 represents -CA. In another preferred embodiments N5 represents T- and N6 represents -CAG.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S3):
wherein
Preferred are the following antisense-oligonucleotides Table 13):
Preferred are also antisense-oligonucleotides of the general formula (S3A) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S3A) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable.
More preferably the antisense-oligonucleotide has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus.
Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 and still more preferable 12 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are GapmeRs of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Thus, preferred are also antisense-oligonucleotides of general formula (S3A)
wherein
Further preferred are antisense-oligonucleotides of general formula (S3A)
wherein
In preferred embodiments N5A represents TTG- and N6A represents -A. In another preferred embodiments N5A represents TG- and N6A represents -AG. In another preferred embodiments N5A represents G- and N6A represents -AGA.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S3A):
wherein N5A represents TG- and N6A represents -AG.
Preferred are the following antisense-oligonucleotides Table 14):
Preferred are also antisense-oligonucleotides of the general formula (S3B) represented by the following sequence:
wherein
Preferably the antisense-oligonucleotide of general formula (S3A) has between 10 and 28 nucleotides and at least one LNA nucleotide at the 3′ terminus and at least one LNA nucleotide at the 5′ terminus. Preferred are also antisense-oligonucleotides having between 10 and 28 nucleotides and two LNA nucleotides at the 3′ terminus and two LNA nucleotides at the 5′ terminus. As LNA nucleotides (LNA units) especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably in the chapter “Preferred LNAs” are suitable and as internucleotides bridges especially these disclosed in the chapter “Internucleotide Linkages (IL)” are suitable.
More preferably the antisense-oligonucleotide has between 11 and 24 nucleotides and at least two LNA nucleotides at the 3′ terminus and at least two LNA nucleotides at the 5′ terminus.
Still more preferably the antisense-oligonucleotide has between 10 and 16, more preferably between 12 and 14 and still more preferable 12 nucleotides and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 3′ terminal end and between 1 and 5, preferably 2 and 4 and more preferably between 2 and 3 LNA units at the 5′ terminal end. Preferably the antisense-oligonucleotides are GapmeRs of the form LNA segment A—DNA segment—LNA segment B. Preferably the antisense-oligonucleotides contain at least 6, more preferably at least 7 and most preferably at least 8 non-LNA units such as DNA units in between the two LNA segments. Suitable nucleobases for the non-LNA units and the LNA units are disclosed in the chapter “Nucleobases”.
Thus, preferred are also antisense-oligonucleotides of general formula (S3B)
wherein
Further preferred are antisense-oligonucleotides of general formula (S3B)
wherein
In preferred embodiments N5B represents TGC- and N6B represents -G. In another preferred embodiments N5B represents GC- and N6B represents -GA. In another preferred embodiments N5B represents C- and N6B represents -GAT.
Preferably, the present invention relates to antisense-oligonucleotides of the formula (S3B):
wherein N5B represents: TGC- and N6B represents -G.
Preferred are the following antisense-oligonucleotides Table 15):
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N5-GCAAAATT-N6-3′ (Seq. ID No. 94) or 5′-N5A-CAAAATTC-N5A-3′ (Seq. ID No. 95) or 5′-N5B-AAAATTCA-N6E-3′ (Seq. ID No. 96), wherein
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 28 nucleotides, wherein at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N5-GCAAAATT-N6-3′ (Seq. ID No. 94) or 5′-N5A-CAAAATTC-N6A-3′ (Seq. ID No. 95) or 5′-N5B-AAAATTCA-N6B-3′ (Seq. ID No. 96), wherein the residues N5, N6, N5A, N6A, N5B and N6B have the meanings especially the further limited meanings as disclosed herein and salts and optical isomers of said antisense-oligonucleotide.
Surprisingly, it has been found that antisense-oligonucleotides consisting of 10 to 16 nucleotides, preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides, wherein at least two of the 10 to 16 nucleotides, preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides, are LNAs, have the advantage that any reagent for transfection like lipofectamine has to be used and “free uptake” is possible.
The present invention preferably relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TGCAAAATTC (Seq. ID No. 71), GCAAAATTCA (Seq. ID No. 72), CAAAATTCAG (Seq. ID No. 73), TGCAAAATTCAG (Seq. ID No. 74), ATTGCAAAATTC (Seq. ID No. 77), TTGCAAAATTCA (Seq. ID No. 78), ACATTGCAAAATTCA (Seq. ID No. 79), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), TTGCAAAATTC (Seq. ID No. 84), TGCAAAATTCA (Seq. ID No. 85), GCAAAATTCAG (Seq. ID No. 86), CATTGCAAAATTC (Seq. ID No. 88), ATTGCAAAATTCA (Seq. ID No. 89), TTGCAAAATTCAG (Seq. ID No. 90), ACATTGCAAAATTC (Seq. ID No. 91), CATTGCAAAATTCA (Seq. ID No. 92), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TGCAAAATTC (Seq. ID No. 71), GCAAAATTCA (Seq. ID No. 72), CAAAATTCAG (Seq. ID No. 73), TGCAAAATTCAG (Seq. ID No. 74), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), TGCAAAATTCA (Seq. ID No. 85), GCAAAATTCAG (Seq. ID No. 86), TTGCAAAATTCAG (Seq. ID No. 90), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TGCAAAATTCAG (Seq. ID No. 74), ATTGCAAAATTC (Seq. ID No. 77), TTGCAAAATTCA (Seq. ID No. 78), ACATTGCAAAATTCA (Seq. ID No. 79), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), CATTGCAAAATTC (Seq. ID No. 88), ATTGCAAAATTCA (Seq. ID No. 89), TTGCAAAATTCAG (Seq. ID No. 90), ACATTGCAAAATTC (Seq. ID No. 91), CATTGCAAAATTCA (Seq. ID No. 92), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TGCAAAATTCAG (Seq. ID No. 74), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), TTGCAAAATTCAG (Seq. ID No. 90), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TGCAAAATTCAG (Seq. ID No. 74), ATTGCAAAATTC (Seq. ID No. 77), TTGCAAAATTCA (Seq. ID No. 78), CATTGCAAAATTC (Seq. ID No. 88), ATTGCAAAATTCA (Seq. ID No. 89), TTGCAAAATTCAG (Seq. ID No. 90), ACATTGCAAAATTC (Seq. ID No. 91), CATTGCAAAATTCA (Seq. ID No. 92), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence TGCAAAATTCAG (Seq. ID No. 74), TTGCAAAATTCAG (Seq. ID No. 90), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
The antisense-oligonucleotides of formula S3, S3A, S3B in form of gapmers (LNA segment 1—DNA segment—LNA segment 2) contain an LNA segment at the 5′ terminal end consisting of 2 to 5, preferably 2 to 4 LNA units and contain an LNA segment at the 3′ terminal end consisting of 2 to 5, preferably 2 to 4 LNA units and between the two LNA segments one DNA segment consisting of 6 to 14, preferably 7 to 12 and more preferably 8 to 11 DNA units.
The antisense-oligonucleotides of formula S3, S3A, S3B contain the LNA nucleotides (LNA units) as disclosed herein, especially these disclosed in the chapter “Locked Nucleic Acids (LNA®)” and preferably these disclosed in the chapter “Preferred LNAs”. The LNA units and the DNA units may comprise standard nucleobases such as adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), but may also contain modified nucleobases as disclosed in the chapter “Nucleobases”. The antisense-oligonucleotides of formula S3, S3A, S3B or the LNA segments and the DNA segment of the antisense-oligonucleotide may contain any internucleotide linkage as disclosed herein and especially these disclosed in the chapter “Internucleotide Linkages (IL)”. The antisense-oligonucleotides of formula S3, S3A, S3B may optionally also contain endgroups at the 3′ terminal end and/or the 5′ terminal end and especially these disclosed in the chapter “Terminal groups”.
Experiments have shown that modified nucleobases do not considerably increase or change the activity of the inventive antisense-oligonucleotides in regard to tested neurological and oncological indications. The modified nucleobases 5-methylcytosine or 2-aminoadenine have been demonstrated to further increase the activity of the antisense-oligonucleotides of formula S3, S3A, S3B especially if 5-methylcytosine is used in the LNA nucleotides only or in the LNA nucleotides and in the DNA nucleotides and/or if 2-aminoadenine is used in the DNA nucleotides and not in the LNA nucleotides.
As LNA units for the antisense-oligonucleotides of formula S3, S3A, S3B especially β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-ENA (b5), β-D-(NH)-LNA (b6), β-D-(NCH3)-LNA (b7), β-D-(ONH)-LNA (b8) and β-D-(ONCH3)-LNA (b9) are preferred. Experiments have been shown that all of these LNA units b1, b2, b4, b5, b6, b7, b8, and b9 can be synthesized with the required effort and lead to antisense-oligonucleotides of comparable stability and activity. However based on the experiments the LNA units b1, b2, b4, b5, b6, and b7 are further preferred. Still further preferred are the LNA units b1, b2, b4, b6, and b7, and even more preferred are the LNA units b1 and b4 and most preferred also in regard to the complexity of the chemical synthesis is the β-D-oxy-LNA (b1).
So far no special 3′ terminal group or 5′ terminal group could be found which remarkably had changed or increased the stability or activity for oncological or neurological indications, so that 3′ and 5′ end groups are possible but not explicitly preferred.
Various internucleotide bridges or internucleotide linkages are possible. In the formulae disclosed herein the internucleotide linkage IL is represented by -IL′-Y—.
Thus, IL=-IL′-Y—=—X″—P(═X′)(X−)—Y—, wherein IL is preferably selected form the group consisting of:
Preferred are the internucleotide linkages IL selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —S—P(O)(O−)—O—, —S—P(O)(S−)—O—, —O—P(O)(O−)—S—, —O—P(O)(S−)—S—, —S—P(O)(O−)—S—, —O—P(O)(OCH3)—O—, —O—P(O)(NH(CH3))—O—, —O—P(O)[N(CH3)2]—O—, —O—P(O)(OCH2CH2OCH3)—O—, and more preferred selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —S—P(O)(O−)—O—, —S—P(O)(S−)—O—, —O—P(O)(O−)—S—, —O—P(O)(S−)—S—, —S—P(O)(O−)—S—, and still more preferred selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, and most preferably selected from —O—P(O)(O−)—O—and —O—P(O)(S−)—O—.
Thus, the present invention preferably relates to an antisense-oligonucleotide in form of a gapmer consisting of 10 to 28 nucleotides, preferably 10 to 18 nucleotides, more preferably 10 to 16, and still more preferably 12 to 16 or 12 to 14 nucleotides and 1 to 5 of these nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the 3′ terminal end of the antisense-oligonucleotide are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the 3′ terminal end a sequence of at least 6, preferably 7 and more preferably 8 DNA nucleotides is present, and the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N5-GCAAAATT-N5-3′ (Seq. ID No. 94) or 5′-N5A-CAAAATTC-N6A-3′ (Seq. ID No. 95) or 5′-N5B-AAAATTCA-N6B-3′ (Seq. ID No. 96), wherein the residues N5, N6, N5A, N6A, N5B and N6B have the meanings especially the further limited meanings as disclosed herein, and
Still further preferred, the present invention relates to an antisense-oligonucleotide in form of a gapmer consisting of 10 to 28 nucleotides, preferably 10 to 18 nucleotides, more preferably 10 to 16, and still more preferably 12 to 16 or 12 to 14 nucleotides and 1 to 5 of these nucleotides at the 5′ terminal end and 1 to 5 nucleotides at the 3′ terminal end of the antisense-oligonucleotide are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the 3′ terminal end a sequence of at least 6, preferably 7 and more preferably 8 DNA nucleotides is present, and the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence 5′-N5-GCAAAATT-N5-3′ (Seq. ID No. 94) or 5′-N5A-CAAAATTC-N6A-3′ (Seq. ID No. 95) or 5′-N5B-AAAATTCA-N6B-3′ (Seq. ID No. 96), wherein the residues N5, N6, N5A, N6A, N5B and N6B have the meanings especially the further limited meanings as disclosed herein, and the LNA nucleotides are selected from β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-(NH)-LNA (b6), and β-D-(NCH3)-LNA (b7); and
Especially preferred are the gapmer antisense-oligonucleotides of Table 11 or Table 12 or Tables 13-15 containing a segment of 2 to 5, preferably 2 to 4 and more preferably 2 to 3 LNA units at the 3′ terminus and a segment of 2 to 5, preferably 2 to 4 and more preferably 2 to 3 LNA units at the 5′ terminus and a segment of at least 6, preferably 7 and more preferably 8 DNA units between the two segments of LNA units, wherein the LNA units are selected from β-D-oxy-LNA (b1), β-D-thio-LNA (b2), α-L-oxy-LNA (b4), β-D-(NH)-LNA (b6), and β-D-(NCH3)-LNA (b7) and the internucleotide linkages are selected from phosphate, phosphorothioate and phosphorodithioate. Such preferred antisense-oligonucleotides may not contain any modified 3′ and 5′ terminal end or may not contain any 3′ and 5′ terminal group and may as modified nucleobase contain 5-methylcytosine in the LNA units, preferably all the LNA units and/or 2-aminoadenine in some or all DNA units and/or 5-methylcytosine in some or all DNA units.
Preferred are the following antisense-oligonucleotides (Table 16):
The antisense-oligonucleotides as disclosed herein such as the antisense-oligonucleotides of Tables 16 and 17 consist of nucleotides, preferably DNA nucleotides, which are non-LNA units (also named herein non-LNA nucleotides) as well as LNA units (also named herein LNA nucleotides). Although not explicitly indicated, the antisense-oligonucleotides of the sequences Seq. ID No.s 4-93 of Table 16 and 17 comprise 2 to 4 LNA nucleotides (LNA units) at the 3′ terminus and 2 to 4 LNA nucleotides (LNA units) at the 5′ terminus.
That means, as long as not explicitly indicated, the antisense-oligonucleotides of the present invention or as disclosed herein by the letter code A, C, G, T and U may contain any internucleotide linkage, any end group and any nucleobase as disclosed herein. Moreover the antisense-oligonucleotides of the present invention or as disclosed herein are gapmers of any gapmer structure as disclosed herein with at least one LNA unit at the 3′ terminus and at least one LNA unit at the 5′ terminus. Moreover any LNA unit as disclosed herein can be used within the antisense-oligonucleotides of the present invention or as disclosed herein. Thus, for instance, the antisense-oligonucleotide AGGTTAGGGCTGAATT (Seq. ID No. 4) or TACAAGCAAGGCATTT (Seq. ID No. 5) or ACATTGCAAAATTCAG (Seq. ID No. 6) or GGTTAGGGCTGA (Seq. ID No. 16) or CAAGCAAGGCAT (Seq. ID No. 46) or TGCAAAATTCAG (Seq. ID No. 74) contains at least one LNA unit at the 5′ terminus and at least one LNA unit at the 3′ terminus, any nucleobase, any 3′ end group, any 5′ end group, any gapmer structure, and any internucleotide linkage as disclosed herein and covers also salts and optical isomers of that antisense-oligonucleotide.
The use of LNA units is preferred especially at the 3′ terminal and the 5′ terminal end. Thus it is preferred if the last 1-5 nucleotides at the 3′ terminal end and also the last 1-5 nucleotides at the 5′ terminal end especially of the sequences disclosed herein and particularly of Seq. ID No.s 4-93 of Table 16 and 17 are LNA units (also named LNA nucleotides) while in between the 1-5 LNA units at the 3′ and 5′ end 2-14, preferably 3-12, more preferably 4-10, more preferably 5-9, still more preferably 6-8, non-LNA units (also named non-LNA nucleotides) are present. Such kinds of antisense-oligonucleotides are called gapmers and are disclosed in more detail below. More preferred are 2-5 LNA nucleotides at the 3′ end and 2-5 LNA nucleotides at the 5′ end or 1-4 LNA nucleotides at the 3′ end and 1-4 LNA nucleotides at the 5′ end and still more preferred are 2-4 LNA nucleotides at the 3′ end and 2-4 LNA nucleotides at the 5′ end of the antisense-oligonucleotides with a number of preferably 4-10, more preferably 5-9, still more preferably 6-8 non-LNA units in between the LNA units at the 3′ and the 5′ end.
Moreover as internucleotide linkages between the LNA units and between the LNA units and the non-LNA units, the use of phosphorothioates or phosphorodithioates and preferably phosphorothioates is preferred.
Thus further preferred are antisense-oligonucleotides wherein more than 50%, preferably more than 60%, more preferably more than 70%, still more preferably more than 80%, and most preferably more than 90% of the internucleotide linkages are phosphorothioates or phosphates and more preferably phosphorothioate linkages and wherein the last 1-4 or 2-5 nucleotides at the 3′ end are LNA units and the last 1-4 or 2-5 nucleotides at the 5′ end are LNA units and between the LNA units at the ends a sequence of 6-14 nucleotides, preferably 7-12, preferably 8-11, more preferably 8-10 are present which are non-LNA units, preferably DNA units. Moreover it is preferred that these antisense-oligonucleotides in form of gapmers consist in total of 12 to 20, preferably 12 to 18 nucleotides, more preferably 12 to 16 nucleotides, more preferably 12 to 14 nucleotides.
The present invention therefore preferably relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCT (Seq. ID No. 9), GTTAGGGCTG (Seq. ID No. 10), TTAGGGCTGA (Seq. ID No. 11), AGGTTAGGGCTG (Seq. ID No. 15), GGTTAGGGCTGA (Seq. ID No. 16), GTTAGGGCTGAA (Seq. ID No. 17), TTAGGGCTGAAT (Seq. ID No. 18), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), GGTTAGGGCTG (Seq. ID No. 21), GTTAGGGCTGA (Seq. ID No. 22), TTAGGGCTGAA (Seq. ID No. 23), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), GTTAGGGCTGAAT (Seq. ID No. 28), TTAGGGCTGAATT (Seq. ID No. 29), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), GTTAGGGCTGAATT (Seq. ID No. 32), AGGTTAGGGCTGAATT (Seq. ID No. 4), CAAGCAAGGC (Seq. ID No. 39), AAGCAAGGCA (Seq. ID No. 40), AGCAAGGCAT (Seq. ID No. 41), TACAAGCAAGGC (Seq. ID No. 44), ACAAGCAAGGCA (Seq. ID No. 45), CAAGCAAGGCAT (Seq. ID No. 46), AAGCAAGGCATT (Seq. ID No. 47), AGCAAGGCATTT (Seq. ID No. 48), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), ACAAGCAAGGC (Seq. ID No. 52), CAAGCAAGGCA (Seq. ID No. 53), AAGCAAGGCAT (Seq. ID No. 54), AGCAAGGCATT (Seq. ID No. 55), TACAAGCAAGGCA (Seq. ID No. 57), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), AAGCAAGGCATTT (Seq. ID No. 60), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62) CAAGCAAGGCATTT (Seq. ID No. 63) TACAAGCAAGGCATTT (Seq. ID No. 5), TGCAAAATTC (Seq. ID No. 71), GCAAAATTCA (Seq. ID No. 72), CAAAATTCAG (Seq. ID No. 73), TGCAAAATTCAG (Seq. ID No. 74), ATTGCAAAATTC (Seq. ID No. 77), TTGCAAAATTCA (Seq. ID No. 78), ACATTGCAAAATTCA (Seq. ID No. 79), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), TTGCAAAATTC (Seq. ID No. 84), TGCAAAATTCA (Seq. ID No. 85), GCAAAATTCAG (Seq. ID No. 86), CATTGCAAAATTC (Seq. ID No. 88), ATTGCAAAATTCA (Seq. ID No. 89), TTGCAAAATTCAG (Seq. ID No. 90), ACATTGCAAAATTC (Seq. ID No. 91), CATTGCAAAATTCA (Seq. ID No. 92), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 10 to 16 nucleotides, wherein at least two of the 10 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCT (Seq. ID No. 9), GTTAGGGCTG (Seq. ID No. 10), TTAGGGCTGA (Seq. ID No. 11), GGTTAGGGCTGA (Seq. ID No. 16), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), GGTTAGGGCTG (Seq. ID No. 21), GTTAGGGCTGA (Seq. ID No. 22), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), AGGTTAGGGCTGAATT (Seq. ID No. 4), CAAGCAAGGC (Seq. ID No. 39), AAGCAAGGCA (Seq. ID No. 40), AGCAAGGCAT (Seq. ID No. 41), CAAGCAAGGCAT (Seq. ID No. 46), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), CAAGCAAGGCA (Seq. ID No. 53), AAGCAAGGCAT (Seq. ID No. 54), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62) CAAGCAAGGCATTT (Seq. ID No. 63) TACAAGCAAGGCATTT (Seq. ID No. 5), TGCAAAATTC (Seq. ID No. 71), GCAAAATTCA (Seq. ID No. 72), CAAAATTCAG (Seq. ID No. 73), TGCAAAATTCAG (Seq. ID No. 74), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), TGCAAAATTCA (Seq. ID No. 85), GCAAAATTCAG (Seq. ID No. 86), TTGCAAAATTCAG (Seq. ID No. 90), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence AGGTTAGGGCTG (Seq. ID No. 15), GGTTAGGGCTGA (Seq. ID No. 16), GTTAGGGCTGAA (Seq. ID No. 17), TTAGGGCTGAAT (Seq. ID No. 18), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), GTTAGGGCTGAAT (Seq. ID No. 28), TTAGGGCTGAATT (Seq. ID No. 29), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), GTTAGGGCTGAATT (Seq. ID No. 32), AGGTTAGGGCTGAATT (Seq. ID No. 4), TACAAGCAAGGC (Seq. ID No. 44), ACAAGCAAGGCA (Seq. ID No. 45), CAAGCAAGGCAT (Seq. ID No. 46), AAGCAAGGCATT (Seq. ID No. 47), AGCAAGGCATTT (Seq. ID No. 48), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), TACAAGCAAGGCA (Seq. ID No. 57), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), AAGCAAGGCATTT (Seq. ID No. 60), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63), TACAAGCAAGGCATTT (Seq. ID No. 5), TGCAAAATTCAG (Seq. ID No. 74), ATTGCAAAATTC (Seq. ID No. 77), TTGCAAAATTCA (Seq. ID No. 78), ACATTGCAAAATTCA (Seq. ID No. 79), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), CATTGCAAAATTC (Seq. ID No. 88), ATTGCAAAATTCA (Seq. ID No. 89), TTGCAAAATTCAG (Seq. ID No. 90), ACATTGCAAAATTC (Seq. ID No. 91), CATTGCAAAATTCA (Seq. ID No. 92), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 16 nucleotides, wherein at least two of the 12 to 16 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCTGA (Seq. ID No. 16), AGGTTAGGGCTGAAT (Seq. ID No. 19), GGTTAGGGCTGAATT (Seq. ID No. 20), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), AGGTTAGGGCTGAATT (Seq. ID No. 4), CAAGCAAGGCAT (Seq. ID No. 46), TACAAGCAAGGCATT (Seq. ID No. 49), ACAAGCAAGGCATTT (Seq. ID No. 50), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63), TACAAGCAAGGCATTT (Seq. ID No. 5), TGCAAAATTCAG (Seq. ID No. 74), CATTGCAAAATTCAG (Seq. ID No. 80), ACATTGCAAAATTCAG (Seq. ID No. 6), TTGCAAAATTCAG (Seq. ID No. 90), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
More preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence AGGTTAGGGCTG (Seq. ID No. 15), GGTTAGGGCTGA (Seq. ID No. 16), GTTAGGGCTGAA (Seq. ID No. 17), TTAGGGCTGAAT (Seq. ID No. 18), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), GTTAGGGCTGAAT (Seq. ID No. 28), TTAGGGCTGAATT (Seq. ID No. 29), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), GTTAGGGCTGAATT (Seq. ID No. 32), TACAAGCAAGGC (Seq. ID No. 44), ACAAGCAAGGCA (Seq. ID No. 45), CAAGCAAGGCAT (Seq. ID No. 46), AAGCAAGGCATT (Seq. ID No. 47), AGCAAGGCATTT (Seq. ID No. 48), TACAAGCAAGGCA (Seq. ID No. 57), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), AAGCAAGGCATTT (Seq. ID No. 60), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63), TGCAAAATTCAG (Seq. ID No. 74), ATTGCAAAATTC (Seq. ID No. 77), TTGCAAAATTCA (Seq. ID No. 78), CATTGCAAAATTC (Seq. ID No. 88), ATTGCAAAATTCA (Seq. ID No. 89), TTGCAAAATTCAG (Seq. ID No. 90), ACATTGCAAAATTC (Seq. ID No. 91), CATTGCAAAATTCA (Seq. ID No. 92), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
Still more preferably, the present invention relates to an antisense-oligonucleotide consisting of 12 to 14 nucleotides, wherein at least two of the 12 to 14 nucleotides are LNAs, wherein the antisense-oligonucleotides is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the antisense-oligonucleotide is represented by the following sequence GGTTAGGGCTGA (Seq. ID No. 16), AGGTTAGGGCTGA (Seq. ID No. 26), GGTTAGGGCTGAA (Seq. ID No. 27), AGGTTAGGGCTGAA (Seq. ID No. 30), GGTTAGGGCTGAAT (Seq. ID No. 31), CAAGCAAGGCAT (Seq. ID No. 46), ACAAGCAAGGCAT (Seq. ID No. 58), CAAGCAAGGCATT (Seq. ID No. 59), TACAAGCAAGGCAT (Seq. ID No. 61), ACAAGCAAGGCATT (Seq. ID No. 62), CAAGCAAGGCATTT (Seq. ID No. 63) TGCAAAATTCAG (Seq. ID No. 74), TTGCAAAATTCAG (Seq. ID No. 90), ATTGCAAAATTCAG (Seq. ID No. 93), and salts and optical isomers of said antisense-oligonucleotide.
It shall be understood, that “coding DNA strand”, as used herein, refers to the DNA strand that is identical to the mRNA (except that is written in the DNA code) and that encompasses the codons that used for protein translation. It is not used as template for the transcription into mRNA. Thus, the terms “coding DNA strand”, “sense DNA strand” and “non-template DNA strand” can be used interchangeably.
Furthermore, “non-coding DNA strand”, as used herein, refers to the DNA strand that is complementary to the “coding DNA strand” and serves as a template for the transcription of mRNA. Thus, the terms “non-coding DNA strand”, “antisense DNA strand” and “template DNA strand” can be used interchangeably
The term “antisense-oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics or variants thereof such. The term “antisense-oligonucleotide” includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleotide (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms, because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. The antisense-oligonucleotides are short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and inhibit its expression.
The term “nucleoside” is well known to a skilled person and refers to a pentose sugar moiety like ribose, desoxyribose or a modified or locked ribose or a modified or locked desoxyribose like the LNAs which are below disclosed in detail. A nucleobase is linked to the glycosidic carbon atom (position 1′ of the pentose) and an internucleotide linkage is formed between the 3′ oxygen or sulfur atom and preferably the 3′ oxygen atom of a nucleoside and the 5′ oxygen or sulfur atom and preferably the 5′ oxygen atom of the adjacent nucleoside, while the internucleotide linkage does not belong to the nucleoside.
The term “nucleotide” is well known to a skilled person and refers to a pentose sugar moiety like ribose, desoxyribose or a modified or locked ribose or a modified or locked desoxyribose like the LNAs which are below disclosed in detail. A nucleobase is linked to the glycosidic carbon atom (position 1′ of the pentose) and an internucleotide linkage is formed between the 3′ oxygen or sulfur atom and preferably the 3′ oxygen atom of a nucleotide and the 5′ oxygen or sulfur atom and preferably the 5′ oxygen atom of the adjacent nucleotide, while the internucleotide linkage is a part of the nucleotide.
The term “nucleobase” is herein abbreviated with “B” and refers to the five standard nucleotide bases adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U) as well as to modifications or analogues thereof or analogues with ability to form Watson-Crick base pair with bases in the complimentary strand. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (C*), 5-hydroxymethyl cytosine, N4-methylcytosine, xanthine, hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 6-ethyladenine, 6-ethylguanine, 2-propyladenine, 2-propylguanine, 6-carboxyuracil, 5-halouracil, 5,6-dihydrouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-aza uracil, 6-aza cytosine, 6-aza thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine, 8-bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-trifluoromethyluracil, 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, 5-iodocytosine, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine etc., with 5-methylcytosine and/or 2-aminoadenine substitutions being preferred since these modifications have been shown to increase nucleic acid duplex stability.
Preferred antisense-oligonucleotides of the present invention can comprise analogues of nucleobases. The nucleobase of only one nucleotide unit of the antisense-oligonucleotide could be replaced by an analogue of a nucleobase or two, three, four, five or even all nucleobases in an antisense-oligonucleotide could be replaced by analogues of nucleobases.
It will be recognized that when referring to a sequence of nucleotides or monomers, what is referred to, is the sequence of bases, such as A, T, G, C or U. The representation of the antisense-oligonucleotides by the letter code A, T, G, C and U has to be understood that said antisense-oligonucleotide may contain any the nucleobases as disclosed herein, any of the 3′ end groups as disclosed herein, any of the 5′ end groups as disclosed herein, and any of the internucleotide linkages (also referred to as internucleotide bridges) as disclosed herein. The nucleotides A, T, G, C and U have also to be understood as being LNA nucleotides or non-LNA nucleotides such as preferably DNA nucleotides.
The antisense-oligonucleotides as well as the salts of the antisense-oligonucleotides as disclosed herein have been proven to be complementary to the target which is the gene encoding for EfnB2 or the mRNA encoding the EfnB2, i.e., hybridize sufficiently well and with sufficient specificity and especially selectivity to give the desired inhibitory effect.
The term “salt” refers to physiologically and/or pharmaceutically acceptable salts of the antisense-oligonucleotides of the present invention. The antisense-oligonucleotides contain nucleobases like adenine, guanine, thymine, cytosine or derivatives thereof which are basic and which form a salt like a chloride or mesylate salt. The internucleotide linkage preferably contains a negatively charged oxygen or sulfur atom which form salts like the sodium, lithium or potassium salt. Thus, pharmaceutically acceptable base addition salts are formed with inorganic bases or organic bases. Examples for suitable organic and inorganic bases are bases derived from metal ions, e.g., aluminum, alkali metal ions, such as sodium or potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion or alkali- or alkaline-earth hydroxides, -carbonates or -bicarbonates. Examples include aqueous LiOH, NaOH, KOH, NH4OH, potassium carbonate, ammonia and sodium bicarbonate, ammonium salts, primary, secondary and tertiary amines, such as, e.g., tetraalkylammonium hydroxide, lower alkylamines such as methylamine, t-butylamine, procaine, ethanolamine, arylalkylamines such as dibenzylamine and N,N-dibenzylethylenediamine, lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines such as cyclohexylamine or dicyclohexylamine, morpholine, glucamine, N-methyl- and N,N-dimethylglucamine, 1-adamantylamine, benzathine, or salts derived from amino acids like arginine, lysine, ornithine or amides of originally neutral or acidic amino acids, chloroprocaine, choline, procaine or the like.
Since the antisense-oligonucleotides are basic, they form pharmaceutically acceptable salts with organic and inorganic acids. Examples of suitable acids for such acid addition salt formation are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p-toluenesulfonic acid, naphthylsulfonic acid, sulfanilic acid, camphersulfonic acid, china acid, mandelic acid, o-methylmandelic acid, hydrogen-benzenesulfonic acid, picric acid, adipic acid, D-o-tolyltartaric acid, tartronic acid, toluic acid, (o, m, p)-toluic acid, naphthylamine sulfonic acid, and other mineral or carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.
In the context of this invention, “hybridization” means nucleic acid hybridization, wherein a single-stranded nucleic acid (DNA or RNA) interacts with another single-stranded nucleic acid having a very similar or even complementary sequence.
Thereby the interaction takes place by hydrogen bonds between specific nucleobases (base pairing).
As used herein, the term “complementarity” (DNA and RNA base pair complementarity) refers to the capacity for precise pairing between two nucleic acids. The nucleotides in a base pair are complementary when their shape allows them to bond together by hydrogen bonds. Thereby forms the pair of adenine and thymidine (or uracil) two hydrogen bonds and the cytosine-guanine pair forms three hydrogen bonds. “Complementary sequences” as used herein means DNA or RNA sequences, being such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things.
The term “specifically hybridizable” as used herein indicates a sufficient degree of complementarity or precise base pairing of the antisense-oligonucleotide to the target sequence such that stable and specific binding occurs between the antisense-oligonucleotide and the DNA or RNA target. The sequence of an -oligonucleotide according to the invention does not need to be 100% complementary to that of its target nucleic acid to be specifically hybridizable, although a 100% complementarity is preferred. Thereby “100% complementarity” means that the antisense-oligonucleotide hybridizes with the target over its complete or full length without mismatch. In other words, within the present invention it is defined that an antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule takes place under physiological or pathological conditions but non-specific binding of the antisense-oligonucleotide to non-target sequences is highly unlikely or even impossible.
Therefore, the present invention refers preferably to antisense oligonucleotides, wherein the antisense oligonucleotides bind with 100% complementarity to the mRNA encoding EfnB2 and do not bind to any other region in the complete human transcriptome. Further preferred the present invention refers to antisense oligonucleotides, wherein the antisense oligonucleotides have 100% complementarity over their complete length to the mRNA encoding EfnB2 and have no off-target effects. Alternatively, the present invention refers preferably to antisense oligonucleotides having 100% complementarity to the mRNA encoding EfnB2 but no complementarity to another mRNA of the human transcriptome. Thereby the term “human transcriptome” refers to the total set of transcripts in the human organism, which means transcripts of all cell types and environmental conditions (at any given time).
The antisense-oligonucleotides of the present invention have in common that they are specific in regard to the region where they bind to the gene or to the mRNA encoding EfnB2. According to the present invention it is preferred that within the human transcriptome, the antisense-oligonucleotides have 100% complementarity over their full length only with the mRNA encoding EfnB2.
The term “mRNA”, as used herein, may encompass both mRNA containing introns (also referred to as pre-mRNA) as well as mature mRNA which does not contain any introns.
The antisense-oligonucleotides of the present invention are able to bind or hybridize with the pre-mRNA and/or with the mature mRNA. That means the antisense-oligonucleotides can bind to or hybridize at an intron region or within an intron region of the Pre-mRNA or can bind to or hybridize at an overlapping intron—exon region of the Pre-mRNA or can bind to or hybridize at an exon region or within an exon region of the Pre-mRNA and the exon region of the mRNA. Preferred are antisense-oligonucleotides which are able to bind to or hybridize with Pre-mRNA and mRNA. Binding or hybridization of the antisense-oligonucleotides (ASO) to the Pre-mRNA inhibits the 5′ cap formation, inhibits splicing of the Pre-mRNA in order to obtain the mRNA and activates RNase H which cleaves the Pre-mRNA. Binding or hybridization of the antisense-oligonucleotides (ASO) to the mRNA activates RNase H which cleaves the mRNA and inhibits binding of the ribosomal subunits.
Preferably, the antisense oligonucleotides according to the present invention bind to or hybridize at an exon region or within an exon region of the Pre-mRNA and the exon region of the mRNA.
The antisense-oligonucleotides of the present invention consist of at least 10 and no more than 28, preferably no more than 24 and more preferably no more than 20 nucleotides and consequently consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, preferably of 10 to 20, or 10 to 19, or 11 to 19, or 12 to 19, or 12 to 18 nucleotides and more preferably of 12 to 16 nucleotides. Preferably at least two, preferably three, more preferably four of these nucleotides are locked nucleic acids (LNA). Shorter antisense-oligonucleotides, i.e. antisense-oligonucleotides having less than 10 nucleotides, are also possible, but the shorter the antisense-oligonucleotides, the higher the risk that the hybridization is not sufficiently strong anymore and that selectivity will decrease or will get lost. Non-selective antisense-oligonucleotides bear the risk to bind to undesired regions in the human transcriptome and to undesired mRNAs coding for other proteins thereby causing undesired side effects. Longer antisense-oligonucleotides having more than 20 nucleotides are also possible but further increasing the length make the synthesis of such antisense-oligonucleotides even more complicated and expensive without any further benefit in increasing selectivity or strength of hybridization or better stability in regard to degradation.
Thus the present invention is directed to antisense-oligonucleotides consisting of 10 to 20 nucleotides. Preferably at least two nucleotides and preferably the 3′ and 5′ terminal nucleotides are LNAs. Thus, it is preferred that at least the terminal 3′ nucleotide is an LNA and also at least the 5′ terminal nucleotide is an LNA. In case more than 2 LNAs are present, it is preferred that the further LNAs are linked to the 3′ or 5′ terminal LNA like it is the case in gapmers as disclosed herein.
One nucleotide building block present in an antisense-oligonucleotide of the present invention can be represented by the following general formula (B1) and (B2):
wherein
Preferably X− represents —O−, —OH, —OCH3, —NH(CH3), —N(CH3)2, —OCH2CH2OCH3, —OCH2CH2SCH3, —BH3−, —CH3, —SH, —SCH3, or —S—; and more preferably —O−, —OH, —OCH3, —N(CH3)2, —OCH2CH2OCH3, —BH3−, —SH, —SCH3, or —S−.
IL′ represents preferably —O—P(O)(O−)—, —O—P(O)(S−)—, —O—P(S)(S−)—, —S—P(O)(O−)—, —S—P(O)(S−)—, —S—P(S)(S−)—, —O—P(O)(O−)—, —O—P(O)(S−)—, —S—P(O)(O−)—, —O—P(O)(RH)—, —O—P(O)(ORH)—, —O—P(O)(NHRH)—, —O—P(O)[N(RH)2]—, —O—P(O)(BH3−)—, —O—P(O)(OCH2CH2ORH)—, —O—P(O)(OCH2CH2SRH)—, —O—P(O)(O−)—, —NRH—P(O)(O−)—, wherein RH is selected from hydrogen and C1-4-alkyl.
The group —O—P(O)(RH)—O— is preferably —O—P(O)(CH3)—O—or —O—P(O)(C2H5)—O—and most preferably —O—P(O)(CH3)—O—.
The group —O—P(O)(ORH)—O— is preferably —O—P(O)(OCH3)—O—or —O—P(O)(OC2H5)—O—and most preferably —O—P(O)(OCH3)—O—.
The group —O—P(O)(NHRH)—O— is preferably —O—P(O)(NHCH3)—O—or —O—P(O)(NHC2H5)—O—and most preferably —O—P(O)(NHCH3)—O—.
The group —O—P(O)[N(RH)2]—O— is preferably —O—P(O)[N(CH3)2]—O— or —O—P(O)[N(C2H5)2]—O— and most preferably —O—P(O)[N(CH3)2]—O—.
The group —O—P(O)(OCH2CH2ORH)—O— is preferably —O—P(O)(OCH2CH2OCH3)—O—or —O—P(O)(OCH2CH2OC2H5)—O—and most preferably —O—P(O)(OCH2CH2OCH3)—O—.
The group —O—P(O)(OCH2CH2SRH)—O— is preferably —O—P(O)(OCH2CH2SCH3)—O—or —O—P(O)(OCH2CH2SC2H5)—O—and most preferably —O—P(O)(OCH2CH2SCH3)—O—.
The group —O—P(O)(O−)—NRH— is preferably —O—P(O)(O−)—NH— or —O—P(O)(O−)—N(CH3)— and most preferably —O—P(O)(O−)—NH—.
The group —NRH—P(O)(O−)—O— is preferably —NH—P(O)(O−)—O—or —N(CH3)—P(O)(O−)—O—and most preferably —NH—P(O)(O−)—O—.
Even more preferably IL′ represents —O—P(O)(O)—, —O—P(O)(S−)—, —O—P(S)(S−)—, —O—P(O)(NHRH)—, or —O—P(O)[N(RH)2]—, and still more preferably IL′ represents —O—P(O)(O−)—, —O—P(O)(S−)—, or —O—P(S)(S−)—, and most preferably IL′ represents —O—P(O)(S−)—, or —O—P(S)(S−)—.
Preferably Y represents —O—.
Preferably B represents a standard nucleobase selected from A, T, G, C, U.
Preferably IL represents —O—P(═O)(S−)— or —O—P(═S)(S−)—.
Thus the following general formula (B3) to (B6) are preferred:
wherein
Examples of preferred nucleotides which are non-LNA units are the following:
The monomers of the antisense-oligonucleotides described herein are coupled together via an internucleotide linkage. Suitably, each monomer is linked to the 3′ adjacent monomer via an internucleotide linkage. The person having ordinary skill in the art would understand that, in the context of the present invention, the 5′ monomer at the end of an oligomer does not comprise a 5′ internucleotide linkage, although it may or may not comprise a 5′ terminal group. The term “internucleotide linkage” is intended to mean a group capable of covalently coupling together two nucleotides, two nucleotide analogues like two LNAs, and a nucleotide and a nucleotide analogue like an LNA. Specific and preferred examples include phosphate groups and phosphorothioate groups.
The nucleotides of the antisense-oligonucleotides of the present invention or contiguous nucleotide sequences thereof are coupled together via internucleotide linkages. Suitably each nucleotide is linked through the 5′ position to the 3′ adjacent nucleotide via an internucleotide linkage.
The antisense-oligonucleotides can be modified by several different ways. Modifications within the backbone are possible and refer to antisense-oligonucleotides wherein the phosphate groups (also named phosphodiester groups) in their internucleotide backbone are partially or completely replaced by other groups. Preferred modified antisense-oligonucleotide backbones include, for instance, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriester, aminoalkylphosphotriesters, methyl, ethyl and C3-C10-alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogues of these, and those having inverted polarity wherein the adjacent pairs of nucleotide units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acids forms thereof are also included and disclosed herein in further detail.
Suitable internucleotide linkages include those listed within WO2007/031091, for example the internucleotide linkages listed on the first paragraph of page 34 of WO2007/031091 (hereby incorporated by reference). It is, in some embodiments, preferred to modify the internucleotide linkage from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate—these two, accepted by RNase H mediated cleavage, also allow that route of antisense inhibition in reducing the expression of the target gene.
The internucleotide linkage consists of the group IL′ which is the group bound to the 3′ carbon atom of the ribose moiety and the group Y which is the group bound to the 5′ carbon atom of the contiguous ribose moiety as shown in the formula (IL′Y) below
The internucleotide linkage IL is represented by -IL′-Y—. IL′ represents —X″—P(═X′)(X−)— so that IL is represented by —X″—P(═X′)(X−)—Y—, wherein the substituents X, X′, X″ and Y have the meanings as disclosed herein.
The internucleotide linkage IL=—X″—P(═X′)(X−)—Y— is preferably selected form the group consisting of:
The group —O—P(O)(RH)—O— is preferably —O—P(O)(CH3)—O—or —O—P(O)(C2H5)—O—and most preferably —O—P(O)(CH3)—O—.
The group —O—P(O)(ORH)—O— is preferably —O—P(O)(OCH3)—O—or —O—P(O)(OC2H5)—O—and most preferably —O—P(O)(OCH3)—O—.
The group —O—P(O)(NHRH)—O— is preferably —O—P(O)(NHCH3)—O—or —O—P(O)(NHC2H5)—O—and most preferably —O—P(O)(NHCH3)—O—.
The group —O—P(O)[N(RH)2]—O— is preferably —O—P(O)[N(CH3)2]—O— or —O—P(O)[N(C2H5)2]—O— and most preferably —O—P(O)[N(CH3)2]—O—.
The group —O—P(O)(OCH2CH2ORH)—O— is preferably —O—P(O)(OCH2CH2OCH3)—O—or —O—P(O)(OCH2CH2OC2H5)—O—and most preferably —O—P(O)(OCH2CH2OCH3)—O—.
The group —O—P(O)(OCH2CH2SRH)—O— is preferably —O—P(O)(OCH2CH2SCH3)—O—or —O—P(O)(OCH2CH2SC2H5)—O—and most preferably —O—P(O)(OCH2CH2SCH3)—O—.
The group —O—P(O)(O−)—NRH— is preferably —O—P(O)(O−)—NH— or —O—P(O)(O−)—N(CH3)— and most preferably —O—P(O)(O−)—NH—.
The group —NRH—P(O)(O−)—O— is preferably —NH—P(O)(O−)—O—or —N(CH3)—P(O)(O−)—O—and most preferably —NH—P(O)(O−)—O—.
Even more preferably IL represents —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —O—P(O)(NHRH)—O—, or —O—P(O)[N(RH)2]—O—, and still more preferably IL represents —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, or —O—P(S)(S−)—O—, and most preferably IL represents —O—P(O)(S−)—O—, or —O—P(O)(O−)—O—.
Thus IL is preferably a phosphate group (—O—P(O)(O−)—O−), a phosphorothioate group (—O—P(O)(S−)—O−) or a phosphorodithioate group (—O—P(S)(S−)—O−).
The nucleotide units or the nucleosides of the antisense-oligonucleotides are connected to each other by internucleotide linkages so that within one antisense-oligonucleotide different internucleotide linkages can be present. The LNA units are preferably linked by internucleotide linkages which are not phosphate groups. The LNA units are linked to each other by a group IL which is preferably selected from —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —O—P(O)(NHRH)—O—, and —O—P(O)[N(RH)2]—O— and more preferably from —O—P(O)(S−)—O—and —O—P(S))(−)—O—.
The non-LNA units are linked to each other by a group IL which is preferably selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —O—P(O)(NHRH)—O—, and —O—P(O)[N(RH)2]—O— and more preferably from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—and —O—P(S)(S−)—O—.
A non-LNA unit is linked to an LNA unit by a group IL which is preferably selected from —O—P(O)(S−)—O—, —O—P(S)(S−)—O—, —O—P(O)(NHRH)—O—, and —O—P(O)[N(RH)2]—O— and more preferably from —O—P(O)(S−)—O—and —O—P(S))(−)—O—.
The term “LNA unit” as used herein refers to a nucleotide which is locked, i.e. to a nucleotide which has a bicyclic structure and especially a bicyclic ribose structure and more especially a bicyclic ribose structure as shown in general formula (II). The bridge “locks” the ribose in the 3′-endo (North) conformation. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon. Alternatively used terms for LNA are bicyclic nucleotides or bridged nucleotides, thus, an alternative term for LNA unit is bicyclic nucleotide unit or bridged nucleotide unit.
The term “non-LNA unit” as used herein refers to a nucleotide which is not locked, i.e. to a nucleotide which has no bicyclic sugar moiety and especially no bicyclic ribose structure and more especially no bicyclic ribose structure as shown in general formula (II). The non-LNA units are most preferably DNA units.
The term “DNA unit” as used herein refers to a nucleotide containing a 2-deoxyribose as sugar. Thus, the nucleotide is made of a nucleobase and a 2-deoxyribose.
The term “unit” as used herein refers to a part or a fragment or a moiety of an antisense-oligonucleotide of the present invention. Thus a “unit” is not a complete molecule, it is a part or a fragment or a moiety of an antisense-oligonucleotide which has at least one position for a covalent linkage to another part or fragment or moiety of the antisense-oligonucleotide. For example, the general structures (B1) to (B6) are units, because they can be covalently linked through the group Y and IL′ or —O— and —O—P(O)(S−)— respectively. Preferably a unit is a moiety consisting of a pentose structure, a nucleobase connected to the pentose structure a 5′ radical group and an IL′ radical group.
The term “building block” or “monomer” as used herein refers to a molecule and especially to a nucleoside which is used in the synthesis of an antisense-oligonucleotide of the present invention.
Examples are the LNA molecules of general formula (I), wherein Y represents a 5′-terminal group and IL′ represents a 3′-terminal group.
Furthermore, pure diastereomeric anti-sense-oligonucleotides are preferred. Preferred are Sp- and Rp-diastereomers as shown at hand-right side:
Suitable sulphur (S) containing internucleotide linkages as provided herein are preferred.
Preferred are phosphorothioate moieties in the backbone where at least 50% of the internucleotide linkages are phosphorothioate groups. Also preferred is that the LNA units, if present, are linked through phosphorothioates as internucleotide linkages. Most preferred is a complete phosphorothioate backbone, i.e. most preferred is when all nucleotide units and also the LNA units (if present) are linked to each other through phosphorothioate groups which are defined as follows: —O—P(O)(S−)—O—which is synonymous to —O—P(O,S)—O—or to —O—P(O−)(S)—O—.
In case the antisense-oligonucleotide is a gapmer, it is preferred that the LNA regions have internucleotide linkages selected from —O—P(O)(S−)—O—and —O—P(S)(S−)—O—and that the non-LNA region, the middle part, has internucleotide linkages selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—and —O—P(S)(S−)—O—and that the LNA regions are connected to the non-LNA region through internucleotide linkages selected from —O—P(O)(O−)—O—, —O—P(O)(S−)—O—and —O—P(S))(−)—O—.
It is even more preferred if all internucleotide linkages which are 9 in a 10-mer and 19 in a 20-mer are selected from —O—P(O)(S−)—O—and —O—P(S)(S−)—O—. Still more preferred is that all internucleotide linkages are phosphorothioate groups (—O—P(O)(S−)—O−) or are phosphorodithioate groups (—O—P(S)(S−)—O−).
It is especially preferred that some of the nucleotides of the general formula (B1) or (B2) in the antisense-oligonucleotides are replaced by so-called LNAs (Locked Nucleic Acids). The abbreviation LNA is a registered trademark, but herein the term “LNA” is solely used in a descriptive manner.
Preferably the terminal nucleotides are replaced by LNAs and more preferred the last 1 to 4 nucleotides at the 3′ end and/or the last 1 to 4 nucleotides at the 5′ end are replaced by LNAs. It is also preferred to have at least the terminal nucleotide at the 3′ end and at the 5′ end replaced by an LNA each.
The term “LNA” as used herein, refers to a bicyclic nucleotide analogue, known as “Locked Nucleic Acid”. It may refer to an LNA monomer, or, when used in the context of an “LNA antisense-oligonucleotide” or an “antisense-oligonucleotide containing LNAs”, LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues. LNA nucleotides are characterized by the presence of a linker group (such as a bridge) between C2′ and C4′ of the ribose sugar ring—for example as shown as the biradical R#—R as described below. The LNA used in the antisense-oligonucleotides of the present invention preferably has the structure of the general formula (I)
wherein for all chiral centers, asymmetric groups may be found in either R or S orientation;
wherein X is selected from —O—, —S—, —N(RN)—, —C(R6R7)—, and preferably X is —O—;
R# and R together represent a bivalent linker group consisting of 1-4 groups or atoms selected from —C(RaRb)—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —O—, —Si(Ra)2—, —S—, —SO2—, —N(Rc)—, and >C═Z, wherein Z is selected from —O—, —S—, and —N(Ra)—, and Ra, Rb and Rc are independently of each other selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkynyl, hydroxy, optionally substituted C1-12-alkoxy, C1-6-alkoxy-C1-6-alkyl, C2-6-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkylenyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkylenyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may represent optionally substituted methylene (═CH2), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation, and;
In preferred embodiments, R# and R together represent a biradical consisting of a groups selected from the group consisting of —C(RaRb)—C(RaRb)—, —C(RaRb)—O—, —C(RaRb)—NRc—, —C(RaRb)—S—, and —C(RaRb)—C(RaRb)—O—, wherein each Ra, Rb and Rc may optionally be independently selected.
In some embodiments, Ra and Rb may be, optionally independently selected from the group consisting of hydrogen and C1-6-alkyl, such as methyl, and preferred is hydrogen.
In preferred embodiments, R1, R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation.
In preferred embodiments R1, R2, R3, R4, and R5 are hydrogen.
In some embodiments, R1, R2, and R3, are independently selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation. In preferred embodiments R1, R2, and R3 are hydrogen.
In preferred embodiments, R4 and R5 are each independently selected from the group consisting of —H, —CH3, —CH2—CH3, —CH2—O—CH3, and —CH═CH2. Suitably in some embodiments, either R4 or R5 are hydrogen, whereas the other group (R4 or R5 respectively) is selected from the group consisting of C1-6-alkyl, C2-6-alkenyl, C2-6-alkynyl, substituted C1-6-alkyl, substituted C2-6-alkenyl, substituted C2-6-alkynyl or substituted acyl (—C(═O)—); wherein each substituted group is mono or poly substituted with substituent groups independently selected from halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl, substituted C2-6-alkynyl, —OJ1, —SJ1, —NJ1J2, —N3, -COOJ1, —CN, —O—C(═O)NJ1J2, —N(H)C(═NH)NJ1J2 or —N(H)C(═X)N(H)J2, wherein X is O or S; and each J1 and J2 is, independently —H, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl, substituted C2-6-alkynyl, C1-6-aminoalkyl, substituted C1-6-aminoalkyl or a protecting group. In some embodiments either R4 or R5 is substituted C1-6-alkyl. In some embodiments either R4 or R5 is substituted methylene, wherein preferred substituent groups include one or more groups independently selected from —F, —NJ1J2, —N3, —CN, —OJ1, —SJ1, —O—C(═O)NJ1J2, —N(H)C(═NH)NJ1J2 or —N(H)C(═O)N(H)J2. In some embodiments each J1 and J2 is, independently —H or C1-6-alkyl. In some embodiments either R4 or R5 is methyl, ethyl or methoxymethyl. In some embodiments either R4 or R5 is methyl. In a further embodiment either R4 or R5 is ethylenyl. In some embodiments either R4 or R5 is substituted acyl. In some embodiments either R4 or R5 is —O—C(═O)NJ1J2. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such 5′ modified bicyclic nucleotides are disclosed in WO 2007/134181 A, which is hereby incorporated by reference in its entirety.
In some embodiments B is a nucleobase, including nucleobase analogues and naturally occurring nucleobases, such as a purine or pyrimidine, or a substituted purine or substituted pyrimidine, such as a nucleobase referred to herein, such as a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil, and/or a modified or substituted nucleobase, such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2′thio-thymine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6-diaminopurine.
In preferred embodiments, R# and R together represent a biradical selected from —C(RaRb)—O—, —C(RaRb)—C(RcRd)—O—, —C(RaRb)—C(RcRd)—C(ReRf)—O—, —C(RaRb)—O—C(RdRe)—, —C(RaRb)—O—C(RdRe)—O—, —C(RaRb)—C(RdRe)—, —C(RaRb)—C(RcRd)—C(ReRf)—, —C(Ra)═C(Rb)—C(RdRe)—, —C(RaRb)—N(Rc)—, —C(RaRb)—C(RdRe)—N(Rc)—, —C(RaRb)—N(Rc)—O—, —C(RaRb)—S—, and —C(RaRb)—C(RdRe)—S—, wherein Ra, Rb, Rc, Rd, Re, and Rf each is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkynyl, hydroxy, C1-12-alkoxy, C1-6-alkoxy-C1-6-alkyl, C2-6-alkenyloxy, carboxy, C1-12-alkoxycarbonyl C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (═CH2). For all chiral centers, asymmetric groups may be found in either R or S orientation.
In a further embodiment R# and R together designate a biradical (bivalent group) selected from —CH2—O—, —CH2—S—, —CH2—NH—, —CH2—N(CH3)—, —CH2—CH2—O—, —CH2—CH(CH3)—, —CH2—CH2—S—, —CH2—CH2—NH—, —CH2—CH2—CH2—, —CH2—CH2—CH2—O—, —CH2—CH2—CH(CH3)—, —CH═CH—CH2—, —CH2—O—CH2—O—, —CH2—NH—O—, —CH2—N(CH3)—O—, —CH2—O—CH2—, —CH(CH3)—O—, —CH(CH2—O—CH3)—O—, —CH2—CH2—, and —CH═CH—. For all chiral centers, asymmetric groups may be found in either R or S orientation.
In some embodiments, R# and R together designate the biradical —C(RaRb)—N(Rc)—O—, wherein Ra and Rb are independently selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl, such as hydrogen, and; wherein Rc is selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl, and preferably hydrogen.
In preferred embodiments, R# and R together represent the biradical —C(RaRb)—O—C(RdRe)—O—, wherein Ra, Rb, Rd, and Re are independently selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl, and preferably hydrogen.
In preferred embodiments, R# and R form the biradical —CH(Z)—O—, wherein Z is selected from the group consisting of C1-6-alkyl, C2-6-alkenyl, C2-6-alkynyl, substituted C1-6-alkyl, substituted C2-6-alkenyl, substituted C2-6-alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio; and wherein each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, —OJ1, —NJ1J2, —SJ1, —N3, —OC(═X)J1, —OC(═X)NJ1J2, —NJ3C(═X)NJ1J2 and —CN, wherein each J1, J2 and J3 is, independently, —H or C1-6-alkyl, and X is O, S or NJ1. In preferred embodiments Z is C1-6-alkyl or substituted C1-6-alkyl. In further preferred embodiments Z is methyl. In preferred embodiments Z is substituted C1-6-alkyl. In preferred embodiments said substituent group is C1-6-alkoxy. In some embodiments Z is CH3OCH2—. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in U.S. Pat. No. 7,399,845 which is hereby incorporated by reference in its entirety. In preferred embodiments, R1, R2, R3, R4, and R5 are hydrogen. In preferred embodiments, R1, R2, and R3 are hydrogen, and one or both of R4, R5 may be other than hydrogen as referred to above and in WO 2007/134181.
In preferred embodiments, R# and R together represent a biradical which comprise a substituted amino group in the bridge such as the biradical —CH2—N(Rc)—, wherein Rc is C1-12-alkyloxy. In preferred embodiments R# and R together represent a biradical -Cq3q4-NOR—, wherein q3 and q4 are independently selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl; wherein each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, —OJ1, —SJ1, —NJ1J2, -COOJ1, —CN, —OC(═O)NJ1J2, —NH—C(═NH)NJ1J2 or —NH—C(═X)NHJ2, wherein X is O or S; and each of J1 and J2 is, independently, —H, C1-6-alkyl, C2-6-alkenyl, C2-6-alkynyl, C1-6-aminoalkyl or a protecting group. For all chiral centers, asymmetric groups may be found in either R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/150729 which is hereby incorporated by reference in its entirety. In preferred embodiments, R1, R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl or substituted C2-6-alkynyl, C1-6-alkoxyl, substituted C1-6-alkoxyl, acyl, substituted acyl, C1-6-aminoalkyl or substituted C1-6-aminoalkyl. In preferred embodiments, R1, R2, R3, R4, and R5 are hydrogen. In preferred embodiments, R1, R2, and R3 are hydrogen and one or both of R4, R5 may be other than hydrogen as referred to above and in WO 2007/134181.
In preferred embodiments R# and R together represent a biradical (bivalent group) —C(RaRb)—O—, wherein Ra and Rb are each independently halogen, C1-12-alkyl, substituted C1-12-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl, substituted C2-6-alkynyl, C1-12-alkoxy, substituted C1-12-alkoxy, —OJ1, —SJ1, —S(O)J1, —SO2-J1, —NJ1J2, —N3, —CN, —C(═O)OJ1, —C(═O)NJ1J2, —C(═O)J1, —OC(═O)NJ1J2, —NH—C(═NH)NJ1J2, —NH—C(═O)NJ1J2, or, —NH—C(═S)NJ1J2; or Ra and Rb together are ═C(q3)(q4); q3 and q4 are each, independently, —H, halogen, C1-12-alkyl or substituted C1-12-alkyl; each substituted group is, independently, mono or poly substituted with substituent groups independently selected from halogen, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl, substituted C2-6-alkynyl, —OJ1, —SJ1, —NJ1J2, —N3, —CN, —C(═O)OJ1, —C(═O)NJ1J2, —C(═O)J1, —OC(═O)NJ1J2, —NH—C(═O)NJ1J2, or —NH—C(═S)NJ1J2 and; each J1 and J2 is independently, —H, C1-6-alkyl, substituted C1-6-alkyl, C2-6-alkenyl, substituted C2-6-alkenyl, C2-6-alkynyl, substituted C2-6-alkynyl, C1-6-aminoalkyl, substituted C1-6-aminoalkyl or a protecting group. Such compounds are disclosed in WO2009006478A, hereby incorporated in its entirety by reference.
In preferred embodiments, R# and R form the biradical -Q-, wherein Q is —C(q1)(q2)C(q3)(q4)-, —C(q1)=C(q3)-, —C[═C(q1)(q2)]—C(q3)(q4)- or —C(q1) (q2)-C[═C(q3)(q4)]-;
As used herein, the term “C1-C6-alkyl” refers to —CH3, —C2H5, —C3H7, —CH(CH3)2, —C4H9, —CH2—CH(CH3)2, —CH(CH3)—C2H5, —C(CH3)3, —C5H11, —CH(CH3)—C3H7, —CH2—CH(CH3)—C2H5, —CH(CH3)—CH(CH3)2, —C(CH3)2—C2H5, —CH2—C(CH3)3, —CH(C2H5)2, —C2H4—CH(CH3)2, —C6H13, —C3H6—CH(CH3)2, —C2H4—CH(CH3)—C2H5, —CH(CH3)—C4H9, —CH2—CH(CH3)—C3H7, —CH(CH3)—CH2—CH(CH3)2, —CH(CH3)—CH(CH3)—C2H5, —CH2—CH(CH3)—CH(CH3)2, —CH2—C(CH3)2—C2H5, —C(CH3)2—C3H7, —C(CH3)2—CH(CH3)2, —C2H4—C(CH3)3, —CH2—CH(C2H5)2, and —CH(CH3)—C(CH3)3. The term “C1-C6-alkyl” shall also include “C1-C6-cycloalkyl” like cyclo-C3H5, cyclo-C4H7, cyclo-C5H9, and cyclo-C6H11.
Preferred are —CH3, —C2H5, —C3H7, —CH(CH3)2, —C4H9, —CH2—CH(CH3)2, —CH(CH3)—C2H5, —C(CH3)3, and —C5H11. Especially preferred are —CH3, —C2H5, —C3H7, and —CH(CH3)2.
The term “C1-C6-alkyl” shall also include “C1-C6-cycloalkyl” like cyclo-C3H5, cyclo-C4H7, cyclo-C5H9, and cyclo-C6H11.
As used herein, the term “C1-C12-alkyl” refers to C1-C6-alkyl, —C7H15, —C8H17, —C9H19, —C10H21, —C11H23, —C12H25.
As used herein, the term “C1-C6-alkylenyl” refers to —CH2—, —C2H4—, —CH(CH3)—, —C3H6—, —CH2—CH(CH3)—, —CH(CH3)—CH2—, —C(CH3)2—, —C4H8—, —CH2—C(CH3)2—, —C(CH3)2—CH2—, —C2H4—CH(CH3)—, —CH(CH3)—C2H4—, —CH2—CH(CH3)—CH2—, —CH(CH3)—CH(CH3)—, —C5H10—, —CH(CH3)—C3H6—, —CH2—CH(CH3)—C2H4—, —C2H4—CH(CH3)—CH2—, —C3H6—CH(CH3)—, —C2H4—C(CH3)2—, —C(CH3)2—C2H4—, —CH2—C(CH3)2—CH2—, —CH2—CH(CH3)—CH(CH3)—, —CH(CH3)—CH2—CH(CH3)—, —CH(CH3)—CH(CH3)—CH2—, —CH(CH3)—CH(CH3)—CH(CH3)—, —C(CH3)2—C3H6—, —CH2—C(CH3)2—C2H4—, —C2H4—C(CH3)2—CH2—, —C3H6—C(CH3)2—, —CH(CH3)—C4H8—, —C6H12—, —CH2—CH(CH3)—C3H6—, —C2H4—CH(CH3)—C2H4—, —C3H6—CH(CH3)—CH2—, —C4H8—CH(CH3)—, —C2H4—CH(CH3)—CH(CH3)—, —CH2—CH(CH3)—CH(CH3)—CH2—, —CH2—CH(CH3)—CH2—CH(CH3)—, —CH(CH3)—C2H4—CH(CH3)—, —CH(CH3)—CH2—CH(CH3)—CH2—, and —CH(CH3)—CH(CH3)—C2H4—.
As used herein, the term “C2-C6-alkenyl” refers to —CH═CH2, —CH2—CH═CH2, —C(CH3)═CH2, —CH═CH—CH3, —C2H4—CH═CH2, —CH2—CH═CH—CH3, —CH═CH—C2H5, —CH2—C(CH3)═CH2, —CH(CH3)—CH═CH, —CH═C(CH3)2, —C(CH3)═CH—CH3, —CH═CH—CH═CH2, —C3H6—CH═CH2, —C2H4—CH═CH—CH3, —CH2—CH═CH—C2H5, —CH═CH—C3H7, —CH2—CH═CH—CH═CH2, —CH═CH—CH═CH—CH3, —CH═CH—CH2—CH═CH2, —C(CH3)═CH—CH═CH2, —CH═C(CH3)—CH═CH2, —CH═CH—C(CH3)═CH2, —C2H4—C(CH3)═CH2, —CH2—CH(CH3)—CH═CH2, —CH(CH3)—CH2—CH═CH2, —CH2—CH═C(CH3)2, —CH2—C(CH3)═CH—CH3, —CH(CH3)—CH═CH—CH3, —CH═CH—CH(CH3)2, —CH═C(CH3)—C2H5, —C(CH3)═CH—C2H5, —C(CH3)═C(CH3)2, —C(CH3)2—CH═CH2, —CH(CH3)—C(CH3)═CH2, —C(CH3)═CH—CH═CH2, —CH═C(CH3)—CH═CH2, —CH═CH—C(CH3)═CH2, —C4H8—CH═CH2, —C3H6—CH═CH—CH3, —C2H4—CH═CH—C2H5, —CH2—CH═CH—C3H7, —CH═CH—C4H9, —C3H6—C(CH3)═CH2, —C2H4—CH(CH3)—CH═CH2, —CH2—CH(CH3)—CH2—CH═CH2, —CH(CH3)—C2H4—CH═CH2, —C2H4—CH═C(CH3)2, —C2H4—C(CH3)═CH—CH3, —CH2—CH(CH3)—CH═CH—CH3, —CH(CH3)—CH2—CH═CH—CH3, —CH2—CH═CH—CH(CH3)2, —CH2—CH═C(CH3)—C2H5, —CH2—C(CH3)═CH—C2H5, —CH(CH3)—CH═CH—C2H5, —CH═CH—CH2—CH(CH3)2, —CH═CH—CH(CH3)—C2H5, —CH═C(CH3)—C3H7, —C(CH3)═CH—C3H7, —CH2—CH(CH3)—C(CH3)═CH2, —CH(CH3)—CH2—C(CH3)═CH2, —CH(CH3)—CH(CH3)—CH═CH2, —CH2—C(CH3)2—CH═CH2, —C(CH3)2—CH2—CH═CH2, —CH2—C(CH3)═C(CH3)2, —CH(CH3)—CH═C(CH3)2, —C(CH3)2—CH═CH—CH3, —CH(CH3)—C(CH3)═CH—CH3, —CH═C(CH3)—CH(CH3)2, —C(CH3)═CH—CH(CH3)2, —C(CH3)═C(CH3)—C2H5, —CH═CH—C(CH3)3, —C(CH3)2—C(CH3)═CH2, —CH(C2H5)—C(CH3)═CH2, —C(CH3)(C2H5)—CH═CH2, —CH(CH3)—C(C2H5)═CH2, —CH2—C(C3H7)═CH2, —CH2—C(C2H5)═CH—CH3, —CH(C2H5)—CH═CH—CH3, —C(C4H9)═CH2, —C(C3H7)═CH—CH3, —C(C2H5)═CH—C2H5, —C(C2H5)═C(CH3)2, —C[C(CH3)3]═CH2, —C[CH(CH3)(C2H5)]═CH2, —C[CH2—CH(CH3)2]═CH2, —C2H4—CH═CH—CH═CH2, —CH2—CH═CH—CH2—CH═CH2, —CH═CH—C2H4—CH═CH2, —CH2—CH═CH—CH═CH—CH3, —CH═CH—CH2—CH═CH—CH3, —CH═CH—CH═CH—C2H5, —CH2—CH═CH—C(CH3)═CH2, —CH2—CH═C(CH3)—CH═CH2, —CH2—C(CH3)═CH—CH═CH2, —CH(CH3)—CH═CH—CH═CH2, —CH═CH—CH2—C(CH3)═CH2, —CH═CH—CH(CH3)—CH═CH2, —CH═C(CH3)—CH2—CH═CH2, —C(CH3)═CH—CH2—CH═CH2, —CH═CH—CH═C(CH3)2, —CH═CH—C(CH3)═CH—CH3, —CH═C(CH3)—CH═CH—CH3, —C(CH3)═CH—CH═CH—CH3, —CH═C(CH3)—C(CH3)═CH2, —C(CH3)═CH—C(CH3)═CH2, —C(CH3)═C(CH3)—CH═CH2, and —CH═CH—CH═CH—CH═CH2.
Preferred are —CH═CH2, —CH2—CH═CH2, —C(CH3)═CH2, —CH═CH—CH3, —C2H4—CH═CH2, —CH2—CH═CH—CH3. Especially preferred are —CH═CH2, —CH2—CH═CH2, and —CH═CH—CH3.
As used herein, the term “C2-C6-alkynyl” refers to —C≡CH, —C≡C—CH3, —CH2—C≡CH, —C2H4—C≡CH, —CH2—C≡C—CH3, —C≡C—C2H5, —C3H6—C≡CH, —C2H4—C≡C—CH3, —CH2—C≡C—C2H5, —C≡C—C3H7, —CH(CH3)—C≡CH, —CH2—CH(CH3)—C≡CH, —CH(CH3)—CH2—C≡CH, —CH(CH3)—C≡C—CH3, —C4H8—C≡CH, —C3H6—C≡C—CH3, —C2H4—C≡C—C2H5, —CH2—C≡C—C3H7, —C≡C—C4H9, —C2H4—CH(CH3)—C≡CH, —CH2—CH(CH3)—CH2—C≡CH, —CH(CH3)—C2H4—C≡CH, —CH2—CH(CH3)—C≡C—CH3, —CH(CH3)—CH2—C≡C—CH3, —CH(CH3)—C≡C—C2H5, —CH2—C≡C—CH(CH3)2, —C≡C—CH(CH3)—C2H5, —C≡C—CH2—CH(CH3)2, —C≡C—C(CH3)3, —CH(C2H5)—C≡C—CH3, —C(CH3)2—C≡C—CH3, —CH(C2H5)—CH2—C≡CH, —CH2—CH(C2H5)—C≡CH, —C(CH3)2—CH2—C≡CH, —CH2—C(CH3)2—C≡CH, —CH(CH3)—CH(CH3)—C≡CH, —CH(C3H7)—C≡CH, —C(CH3)(C2H5)—C≡CH, —C≡C—C≡CH, —CH2—C≡C—C≡CH, —C≡C—C≡C—CH3, —CH(C≡CH)2, —C2H4—C≡C—C≡CH, —CH2—C≡C—CH2—C≡CH, —C≡C—C2H4—C≡CH, —CH2—C≡C—C≡C—CH3, —C≡C—CH2—C≡C—CH3, —C≡C—C≡C—C2H5, —C≡C—CH(CH3)—C≡CH, —CH(CH3)—C≡C—C≡CH, —CH(C≡CH)—CH2—C≡CH, —C(C≡CH)2—CH3, —CH2—CH(C≡CH)2, —CH(C≡CH)—C≡C—CH3. Preferred are —C≡CH and —C≡C—CH3.
The term “C1-6-alkoxyl” refers to “C1-C6-alkyl-O—”. The term “C1-12-alkoxyl” refers to “C1-C12-alkyl-O—”. The term “C1-6-aminoalkyl” refers to “H2N—C1-C6-alkyl-”. The term “C2-C6-alkenyloxy” refers to “C2-C6-alkenyl-O—”. The term “C1-6-alkylcarbonyl” refers to “C1-C6-alkyl-CO—”. Also referred to as “acyl”. The term “C1-12-alkylcarbonyl” refers to “C1-C12-alkyl-CO—”. Also referred to as “acyl”. The term “C1-6-alkoxycarbonyl” refers to “C1-C6-alkyl-O—CO—”. The term “C1-12-alkoxycarbonyl” refers to “C1-C12-alkyl-O—CO—”. The term “C1-C6-alkanoyloxy” refers to “C1-C6-alkyl-CO—O—”. The term “C1-6-alkylthio” refers to “C1-C6-alkyl-S—”. The term “C1-6-alkylsulphonyloxy” refers to “C1-C6-alkyl-SO2—O—”. The term “C1-6-alkylcarbonylamino” refers to “C1-C6-alkyl-CO—NH—”. The term “C1-6-alkylamino” refers to “C1-C6-alkyl-NH—”.
The term “(C1-6-)2alkylamino” refers to a dialkylamino group like “[C1-C6-alkyl][C1-C6-alkyl]N—”. The term “C1-6-alkylaminocarbonyl” refers to “C1-C6-alkyl-NH—CO—” The term “(C1-6-)2alkylaminocarbonyl” refers to a dialkylaminocarbonyl group like “[C1-C6-alkyl][C1-C6-alkyl]N—CO—”. The term “amino-C1-12-alkylaminocarbonyl” refers to “H2N—[C1-C6-alkylenyl]-NH—CO—”. The term “C1-6-alkyl-amino-C1-6-alkylaminocarbonyl” refers to “C1-6-alkyl-HN—[C1-C6-alkylenyl]-NH—CO—”. The term “(C1-6-)2alkyl-amino-C1-6-alkylaminocarbonyl” refers to “[C1-C6-alkyl][C1-C6-alkyl]N—[C1-C6-alkylenyl]-NH—CO—”. The term “aryl” refers to phenyl, toluyl, substituted phenyl and substituted toluyl. The term “aryloxy” refers to “aryl-O—”. The term “arylcarbonyl” refers to “aryl-CO—”. The term “aryloxycarbonyl” refers to “aryl-O—CO—”.
The term “heteroaryl” refers to substituted or not substituted heteroaromatic groups which have from 4 to 9 ring atoms, from 1 to 4 of which are selected from O, N and/or S. Preferred “heteroaryl” groups have 1 or 2 heteroatoms in a 5- or 6-membered aromatic ring. Mono and bicyclic ring systems are included. Typical “heteroaryl” groups are pyridyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, pyridazinyl, pyrimidyl, pyrazinyl, 1,3,5-triazinyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, indolizinyl, indolyl, isoindolyl, benzo[b]furyl, benzo[b]thienyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, quinolizinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, tetrahydroquinolyl, benzooxazolyl, chrom-2-onyl, indazolyl, and the like.
The term “heteroaryloxy” refers to “heteroaryl-O—”. The term “heteroarylcarbonyl” refers to “heteroaryl-CO—”. The term “heteroaryloxycarbonyl” refers to “heteroaryl-O—CO—”.
The term “substituted” refers to groups wherein one or more hydrogen atoms are replaced by one or more of the following substituents: —OH, —OCH3, —OC2H5, —OC3H7, —O-cyclo-C3H5, —OCH(CH3)2, —OCH2Ph, —F, —Cl, —COCH3, —COC2H5, —COC3H7, —CO-cyclo-C3H5, —COCH(CH3)2, —COOH, —CONH2, —NH2, —NHCH3, —NHC2H5, —NHC3H7, —NH-cyclo-C3H5, —NHCH(CH3)2, —N(CH3)2, —N(C2H5)2, —N(C3H7)2, —N(cyclo-C3H5)2, —N[CH(CH3)2]2, —SO3H, —OCF3, —OC2F5, cyclo-C3H5, —CH3, —C2H5, —C3H7, —CH(CH3)2, —CH═CH2, —CH2—CH═CH2, —C≡CH and/or —C≡C—CH3.
In case the general structure (I) represents monomers or building blocks for synthesizing the antisense-oligonucleotides of the present invention, the terminal groups Y and IL′ are selected independently of each other from hydrogen, azido, halogen, cyano, nitro, hydroxy, PG-O-, AG-O-, mercapto, PG-S-, AG-S-, C1-6-alkylthio, amino, PG-N(RH)—, AG-N(RH)—, mono- or di(C1-6-alkyl)amino, optionally substituted C1-6-alkoxy, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkenyloxy, optionally substituted C2-6-alkynyl, optionally substituted C2-6-alkynyloxy, monophosphate, monothiophosphate, diphosphate, dithiophosphate triphosphate, trithiophosphate, carboxy, sulphono, hydroxymethyl, PG-O—CH2—, AG-O—CH2—, aminomethyl, PG-N(RH)—CH2—, AG-N(RH)—CH2—, carboxymethyl, sulphonomethyl, where PG is a protection group for —OH, —SH, and —NH(RH), respectively, AG is an activation group for —OH, —SH, and —NH(RH), respectively, and RH is selected from hydrogen and C1-6-alkyl.
The protection groups PG of hydroxy substituents comprise substituted trityl, such as 4,4′-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), optionally substituted 9-(9-phenyl)xanthenyl (pixyl), optionally substituted methoxytetrahydropyranyl (mthp), silyl such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triethylsilyl, and phenyldimethylsilyl, tert-butylethers, acetals (including two hydroxy groups), acyl such as acetyl or halogen substituted acetyls, e.g. chloroacetyl or fluoroacetyl, isobutyryl, pivaloyl, benzoyl and substituted benzoyls, methoxymethyl (MOM), benzyl ethers or substituted benzyl ethers such as 2,6-dichlorobenzyl (2,6-Cl2Bzl). Alternatively when Y or IL′ is hydroxyl they may be protected by attachment to a solid support optionally through a linker.
When Y or IL′ is an amino group, illustrative examples of the amino protection groups are fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (BOC), trifluoroacetyl, allyloxycarbonyl (alloc or AOC), benzyloxycarbonyl (Z or Cbz), substituted benzyloxycarbonyls such as 2-chloro benzyloxycarbonyl (2-CIZ), monomethoxytrityl (MMT), dimethoxytrityl (DMT), phthaloyl, and 9-(9-phenyl)xanthenyl (pixyl).
Act represents an activation group for —OH, —SH, and —NH(RH), respectively. Such activation groups are, for instance, selected from optionally substituted O-phosphoramidite, optionally substituted O-phosphortriester, optionally substituted O-phosphordiester, optionally substituted H-phosphonate, and optionally substituted O-phosphonate.
In the present context, the term “phosphoramidite” means a group of the formula —P(ORx)—N(Ry)2, wherein Rx designates an optionally substituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and each of Ry designate optionally substituted alkyl groups, e.g. ethyl or isopropyl, or the group —N(Ry)2 forms a morpholino group (—N(CH2CH2)2O). Rx preferably designates 2-cyanoethyl and the two Ry are preferably identical and designate isopropyl. Thus, an especially relevant phosphoramidite is N,N-diisopropyl-O-(2-cyanoethyl)-phosphoramidite.
The LNA monomers or LNA building blocks used as starting materials in the synthesis of the antisense-oligonucleotides of the present invention are preferably LNA nucleosides of the following general formulae:
The LNA building blocks are normally provided as LNA phosphoramidites with the four different nucleobases: adenine (A), guanine (G), 5-methyl-cytosine (C*) and thymine (T). The antisense-oligonucleotides of the present invention containing LNA units are synthesized by standard phosphoramidite chemistry. In the LNA building blocks the nucleobases are protected. A preferred protecting group for the amino group of the purin base is a benzoyl group (Bz), indicated as ABz. A preferred protecting group for the amino group of the 5-methylpyrimidinone base is a benzoyl group (Bz), indicated as C*Bz. A preferred protecting group for the amino group of the purinone base is a dimethylformamidine (DMF) group, a diethylformamidine (DEF), a dipropylformamidine (DPF), a dibutylformamidine (DBF), or a iso-butyryl (—CO—CH(CH3)2) group, indicated as GDMF, GDEF, GDPF, GDBF, or GiBu. Thus the group -NDMF refers to —N═CH—N(CH3)2. DMT refers to 4,4′-dimethoxytrityl. Thus, LNA-T refers to 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite-thymidine LNA. LNA-C*Bz refers to 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-4-N-benzoyl-5-methyl-2′-cytidine LNA. LNA-ABz refers to 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-di-isopropyl)phosphoramidite-6-N-benzoyl-2′-adenosine LNA. LNA-GDMF refers to 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite-2-N-dimethylformamidine-2′-guanosine LNA. LNA-GiBu refers to 5′-O-(4,4′-dimethoxy-trityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-2-N-butyryl-2′-guanosine LNA.
In case Y represents the 5′-terminal group of an antisense-oligonucleotide of the present invention, the residue Y is also named Y5′ and represents:
More preferred are: —OCH3, —OC2H5, —OC3H7, —O-cyclo-C3H5, —OCH(CH3)2, —OC(CH3)3, —OC4H9, —OPh, —OCH2-Ph, —O—COCH3, —O—COC2H5, —O—COC3H7, —O—CO-cyclo-C3H5, —O—COCH(CH3)2, —OCF3, —O—S(O)CH3, —O—S(O)C2H5, —O—S(O)C3H7, —O—S(O)-cyclo-C3H5, —O—SO2CH3, —O—SO2C2H5, —O—SO2C3H7, —O—SO2-cyclo-C3H5, —O—SO2—OCH3, —O—SO2—OC2H5, —O—SO2—OC3H7, —O—SO2—O-cyclo-C3H5, —O(CH2)nN[(CH2)nOH], —O(CH2)nN[(CH2)n—H], —O—P(O)(O−)OC3H6OH, —O—P(O)(S−)OC3H6OH, even more preferred are:
In case IL′ represents the 3′-terminal group of an antisense-oligonucleotide of the present invention, the residue IL′ is also named IL′3′ and represents:
More preferred are: —OCH3, —OC2H5, —OC3H7, —O-cyclo-C3H5, —OCH(CH3)2, —OC(CH3)3, —OC4H9, —OPh, —OCH2-Ph, —O—COCH3, —O—COC2H5, —O—COC3H7, —O—CO-cyclo-C3H5, —O—COCH(CH3)2, —OCF3, —O—S(O)CH3, —O—S(O)C2H5, —O—S(O)C3H7, —O—S(O)-cyclo-C3H5, —O—SO2CH3, —O—SO2C2H5, —O—SO2C3H7, —O—SO2-cyclo-C3H5, —O—SO2—OCH3, —O—SO2—OC2H5, —O—SO2—OC3H7, —O—SO2—O-cyclo-C3H5, —O(CH2)nN[(CH2)nOH], —O(CH2)nN[(CH2)n—H], —O—P(O)(O−)OC3H6OH, —O—P(O)(S−)OC3H6OH, even more preferred are:
In preferred embodiments LNA units used in the antisense-oligonucleotides of the present invention preferably have the structure of general formula (II):
The moiety —C(RaRb)—X— represents preferably —C(RaRb)—O—, —C(RaRb)—NRc—, —C(RaRb)—S—, and —C(RaRb)—C(RaRb)—O—, wherein the substituents Ra, Rb and Rc have the meanings as defined herein and are preferably C1-6-alkyl and more preferably C1-4-alkyl. More preferably —C(RaRb)—X— is selected from —CH2—O—, —CH2—S—, —CH2—NH—, —CH2—N(CH3)—, —CH2—CH2—O—, or —CH2—CH2—S—, and more preferably —CH2—O—, —CH2—S—, —CH2—CH2—O—, or —CH2—CH2—S—, and still more preferably —CH2—O—, —CH2—S—, or —CH2—CH2—O—, and still more preferably —CH2—O— or —CH2—S—, and most preferably —CH2—O—.
All chiral centers and asymmetric substituents (if any) can be either in R or in S orientation. For example, two exemplary stereochemical isomers are the beta-D and alpha-L isoforms as shown below:
Preferred LNA units are selected from general formula (b1) to (b9):
The term “thio-LNA” comprises a locked nucleotide in which X in the general formula (II) is selected from —S— or —CH2—S—. Thio-LNA can be in both beta-D and alpha-L-configuration.
The term “amino-LNA” comprises a locked nucleotide in which X in the general formula (II) is selected from —NH—, —N(R)—, —CH2—NH—, and —CH2—N(R)—, where R is selected from hydrogen and C1-4-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.
The term “oxy-LNA” comprises a locked nucleotide in which X in the general formula (II) is —O—. Oxy-LNA can be in both beta-D and alpha-L-configuration.
The term “ENA” comprises a locked nucleotide in which X in the general formula (II) is —CH2—O— (where the oxygen atom of —CH2—O— is attached to the 2′-position relative to the base B). Ra and Rb are independently of each other hydrogen or methyl.
In preferred exemplary embodiments LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.
The antisense-oligonucleotides of the invention may consist of nucleotide sequences which comprise both DNA nucleotides which are non-LNA units as well as LNA nucleotides, and may be arranged in the form of a gapmer. Herein preferred are LNA gapmers having fully phosphorothioated backbones. In general phosphorothioated backbones ensure exceptional resistance to enzymatic degradation.
Thus, the antisense-oligonucleotides of the present invention are preferably gapmers. A gapmer consists of a middle part of DNA nucleotide units which are not locked, thus which are non-LNA units. The DNA nucleotides of this middle part could be linked to each other by the internucleotide linkages (IL) as disclosed herein which preferably may be phosphate groups, phosphorothioate groups or phosphorodithioate groups and which may contain nucleobase analogues such as 5-propynyl cytosine, 7-methylguanine, 7-methyladenine, 2-aminoadenine, 2-thiothymine, 2-thiocytosine, or 5-methylcytosine. That DNA units or DNA nucleotides are not bicyclic pentose structures. The middle part of non-LNA units is flanked at the 3′ end and the 5′ end by sequences consisting of LNA units. Thus gapmers have the general formula:
LNA sequence 1—non-LNA sequence—LNA sequence 2
or
region A—region B—region C
The middle part of the antisense-oligonucleotide which consists of DNA nucleotide units which are non-LNA units is, when formed in a duplex with the complementary target RNA, capable of recruiting RNase. The 3′ and 5′ terminal nucleotide units are LNA units which are preferably in alpha-L configuration, particularly preferred being beta-D-oxy-LNA and alpha-L-oxy LNAs.
Thus, a gapmer is an antisense-oligonucleotide which comprises a contiguous stretch of DNA nucleotides which is capable of recruiting an RNase, such as RNaseH, such as a region of at least 6 or 7 DNA nucleotides which are non-LNA units, referred to herein as middle part or region B, wherein region B is flanked both 5′ and 3′ by regions of affinity enhancing nucleotide analogues which are LNA units, such as between 1-6 LNA units 5′ and 3′ to the contiguous stretch of DNA nucleotides which is capable of recruiting RNase—these flanking regions are referred to as regions A and C respectively.
Preferably the gapmers comprises a (poly)nucleotide sequence of formula (5′ to 3′), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (5′ region) consists of at least one nucleotide analogue, such as at least one LNA unit, such as between 1-6 LNA units, and region B consists of at least five consecutive DNA nucleotides which are non-LNA units and which are capable of recruiting RNase (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), and region C (3′region) consists of at least one nucleotide analogue, such as at least one LNA unit, such as between 1-6 LNA units, and region D, when present consists of 1, 2 or 3 DNA nucleotide units which are non-LNA units.
In some embodiments, region A consists of 1, 2, 3, 4, 5 or 6 LNA units, such as between 2-5 LNA units, such as 2 or 3 LNA units; and/or region C consists of 1, 2, 3, 4, 5 or 6 LNA units, such as between 2-5 LNA units, such as 2 or 3 LNA units.
In some embodiments B consists of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive DNA nucleotides which are capable of recruiting RNase, or between 6-10, or between 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNase. In some embodiments region B consists of at least one DNA nucleotide unit, such as 1-12 DNA nucleotide units, preferably between 4-12 DNA nucleotide units, more preferably between 6-10 DNA nucleotide units, still more preferred such as between 7-10 DNA nucleotide units, and most preferably 8, 9 or 10 DNA nucleotide units which are non-LNA units.
In some embodiments region A consist of 1 to 4 LNA, region B consists of 7, 8, 9, 10 or 11 DNA nucleotide units, and region C consists of 1 to 4 LNA units. Such designs include (A-B-C): 1-7-2, 2-7-1, 2-7-2, 3-7-1, 3-7-2, 1-7-3, 2-7-3, 3-7-3, 2-7-4, 3-7-4, 4-7-2, 4-7-3, 4-7-4, 1-8-1, 1-8-2, 2-8-1, 2-8-2, 1-8-3, 3-8-1, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 3-9-3, 1-9-3, 3-9-1, 4-9-1, 1-9-4, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1, 2-10-2, 2-10-3, 3-10-2, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 1-11-1, 1-11-2, 2-11-1, 2-11-2, 1-11-3, 3-11-1, 2-11-2, 2-11-3, 3-11-2, 3-11-3, 2-11-4, 4-11-2, 3-11-4, 4-11-3, 4-11-4, and may further include region D, which may have one or 2 DNA nucleotide units, which are non-LNA units. Further gapmer designs are disclosed in WO2004/046160A and are hereby incorporated by reference.
In some embodiments the antisense-oligonucleotide consists of a contiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14 nucleotide units (LNA units and non-LNA units together), wherein the contiguous nucleotide sequence is of formula (5′-3′), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein A consists of 1, 2 or 3 LNA units, and B consists of 7, 8 or 9 contiguous DNA nucleotide units which are non-LNA units and which are capable of recruiting RNase when formed in a duplex with a complementary RNA molecule (such as a mRNA target), and C consists of 1, 2 or 3 LNA units. When present, D consists of a single DNA nucleotide unit which is a non-LNA unit.
In some embodiments A consists of 1 LNA unit. In some embodiments A consists of 2 LNA units. In some embodiments A consists of 3 LNA units. In some embodiments C consists of 1 LNA unit. In some embodiments C consists of 2 LNA units. In some embodiments C consists of 3 LNA units. In some embodiments B consists of 7 DNA nucleotide units which are non-LNA units. In some embodiments B consists of 8 DNA nucleotide units which are non-LNA units. In some embodiments B consists of 9 DNA nucleotide units which are non-LNA units. In some embodiments B consists of 1-9 DNA nucleotide units which are non-LNA units, such as 2, 3, 4, 5, 6, 7 or 8 DNA nucleotide units. The DNA nucleotide units are always non-LNA units. In some embodiments B comprises 1, 2 or 3 LNA units which are preferably in the alpha-L configuration and which are more preferably alpha-L-oxy LNA units. In some embodiments the number of nucleotides present in A-B-C are selected from the group consisting of (LNA units—region B—LNA units and more preferably alpha-L-oxy LNA units (region A)-region B—(region C) alpha-L-oxy LNA units): 1-7-2, 2-7-1, 2-7-2, 3-7-1, 3-7-2, 1-7-3, 2-7-3, 3-7-3, 2-7-4, 3-7-4, 4-7-2, 4-7-3, 4-7-4, 1-8-1, 1-8-2, 2-8-1, 2-8-2, 1-8-3, 3-8-1, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 3-9-3, 1-9-3, 3-9-1, 4-9-1, 1-9-4, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1, 2-10-2, 2-10-3, 3-10-2, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 1-11-1, 1-11-2, 2-11-1, 2-11-2, 1-11-3, 3-11-1, 2-11-2, 2-11-3, 3-11-2, 3-11-3, 2-11-4, 4-11-2, 3-11-4, 4-11-3, 4-11-4. In further preferred embodiments the number of nucleotides in A-B-C are selected from the group consisting of: 2-8-2, 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 2-9-2, 3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 2-10-2, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 2-11-2, 2-11-4, 4-11-2, 3-11-4, 4-11-3 and still more preferred are: 2-8-2, 3-8-3, 3-8-4, 4-8-3, 4-8-4, 2-9-2, 3-9-3, 4-9-3, 3-9-4, 4-9-4, 2-10-2, 3-10-3, 3-10-4, 4-10-3, 4-10-4, 2-11-2, 3-11-4, and 4-11-3.
Phosphorothioate, phosphate or phosphorodithioate and especially phosphorothioate internucleotide linkages are also preferred, particularly for the gapmer region B. Phosphorothioate, phosphate or phosphorodithioate linkages and especially phosphorothioate internucleotide linkages may also be used for the flanking regions (A and C, and for linking A or C to D, and within region D, if present).
Regions A, B and C, may however comprise internucleotide linkages other than phosphorothioate or phosphorodithioate, such as phosphodiester linkages, particularly, for instance when the use of nucleotide analogues protects the internucleotide linkages within regions A and C from endo-nuclease degradation—such as when regions A and C consist of LNA units.
The internucleotide linkages in the antisense-oligonucleotide may be phosphodiester, phosphorothioate, phosphorodithioate or boranophosphate so as to allow RNase H cleavage of targeted RNA. Phosphorothioate or phosphorodithioate is preferred, for improved nuclease resistance and other reasons, such as ease of manufacture. In one aspect of the oligomer of the invention, the LNA units and/or the non-LNA units are linked together by means of phosphorothioate groups.
It is recognized that the inclusion of phosphodiester linkages, such as one or two linkages, into an otherwise phosphorothioate antisense-oligonucleotide, particularly between or adjacent to LNA units (typically in region A and or C) can modify the bioavailability and/or bio-distribution of an antisense-oligonucleotide (see WO2008/053314A which is hereby incorporated by reference).
In some embodiments, such as in the sequences of the antisense-oligonucleotides disclosed herein and where suitable and not specifically indicated, all remaining internucleotide linkage groups are either phosphodiester groups or phosphorothioate groups, or a mixture thereof.
In some embodiments all the internucleotide linkage groups are phosphorothioate groups. When referring to specific gapmer antisense-oligonucleotide sequences, such as those provided herein, it will be understood that, in various embodiments, when the linkages are phosphorothioate linkages, alternative linkages, such as those disclosed herein may be used, for example phosphate (also named phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA units. Likewise, when referring to specific gapmer antisense-oligonucleotide sequences, such as those provided herein, when the C residues are annotated as 5′-methyl modified cytosine, in various embodiments, one or more of the Cs present in the oligomer may be unmodified C residues.
As used herein the abbreviations b, d, s, ss have the following meaning:
The following antisense-oligonucleotides in form of gapmers as listed in Table 18 to Table 29 are preferred. The antisense-oligonucleotides as disclosed herein such as the antisense-oligonucleotides of Tables 18 to 29 consist of nucleotides, preferably DNA nucleotides, which are non-LNA units (also named herein non-LNA nucleotides) as well as LNA units (also named herein LNA nucleotides). Although not explicitly indicated, the antisense-oligonucleotides of the sequences Although not explicitly indicated, the “C” in Tables 18 to 29 which refer to LNA units preferably contain 5-methylcytosine (C*) as nucleobase.
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsGbsAb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTbsGb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAb
TbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
TbsAbsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
GbsdGsdTsdTsdAsdGsdGsdGsdCsdTsGbsAb
AbsdGsdGsdTsdTsdAsdGsdGsdGsdCsdTbsGb
GbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAb
TbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
TbsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsddTsGb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb
TbsTbsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb
TbsAbsdGsdGsdGsdCsdTsdGsdAsdAsdTsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsTb
AbsdGsdGsdTsdTsdAsdGsdGsdGsdCbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsGb
GbsdGsdTsdTsdAsdGsdGsdGsdCsTbsGb
GbsdTsdTsdAsdGsdGsdGsdCsdTsGbsAb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAb
TbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAb
TbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAb
TbsAbsdGsdGsdGsdCsdTsdGsdAsdAsTb
TbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
AbsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsdGsdGsdCsdTsdGsdAsdAsdTsTb
AbsdGsdGsdTsdTsdAsdGsdGsdGsdCb
GbsdGsdTsdTsdAsdGsdGsdGsdCsTb
GbsdTsdTsdAsdGsdGsdGsdCsdTsGb
TbsdTsdAsdGsdGsdGsdCsdTsdGsAb
TbsdAsdGsdGsdGsdCsdTsdGsdAsAb
AbsdGsdGsdGsdCsdTsdGsdAsdAsTb
GbsdGsdGsdCsdTsdGsdAsdAsdTsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsGbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
TbsTbsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsGbsAb
GbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAb
GbsTbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
TbsTbsAbsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsCbsTbsGbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsGbsAbsAb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTb
TbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsGbsAbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsGbsAbsAb
GbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTb
GbsTbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsGbsAbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
GbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsGbsAbsAbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTb
GbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTb
GbsGbsTbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTbsTb
AbsGbsGbsTbsTbsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsGbsAbsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbsAbsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbsTbsTb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsCbsAb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsGbsCb
AbsAbsdGsdCsdAsdAsdGsdGsdCsdAsTbsTb
AbsGbsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
CbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
AbsdCsdAsdAsdGsdCsdAsdAsdGsdGsCbsAb
TbsdAsdCsdAsdAsdGsdCsdAsdAsdGsGbsCb
AbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTb
AbsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsCb
AbsAbsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb
AbsGbsdCsdAsdAsdGsdGsdCsdAsdTsdTsTb
TbsdAsdCsdAsdAsdGsdCsdAsdAsGbsGb
AbsdCsdAsdAsdGsdCsdAsdAsdGsGbsCb
CbsdAsdAsdGsdCsdAsdAsdGsdGsCbsAb
AbsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
AbsdGsdCsdAsdAsdGsdGsdCsdAsdTbsTb
GbsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsGb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsCb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsAb
AbsAbsdGsdCsdAsdAsdGsdGsdCsdAsTb
AbsGbsdCsdAsdAsdGsdGsdCsdAsddTsTb
GbsCbsdAsdAsdGsdGsdCsdAsdTsdTsTb
TbsdAsdCsdAsdAsdGsdCsdAsdAsGb
AbsdCsdAsdAsdGsdCsdAsdAsdGsGb
CbsdAsdAsdGsdCsdAsdAsdGsdGsCb
AbsdAsdGsdCsdAsdAsdGsdGsdCsAb
AbsdGsdCsdAsdAsdGsdGsdCsdAsdTb
GbsdCsdAsdAsdGsdGsdCsdAsdTsTb
CbsdAsdAsdGsdGsdCsdAsdTsdTsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsCbsAb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTb
AbsAbsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsCbsAb
AbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
CbsAbsAbsdGsdCsdAsdAsdGsdGsdCsdAsTbsTb
AbsAbsGbsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsGbsCbsAb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsCbsAbsTb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTb
AbsAbsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTb
AbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTb
CbsAbsAbsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsCbsAbsTb
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTb
CbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsCbsAbsTb
AbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTb
CbsAbsAbsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbs
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbs
AbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTbs
AbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTbs
AbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTbsTb
TbsAbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTbsTb
TbsAbsCbsAbsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbsTbsTb
TbsAbsCbsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbsTbsTbsTb
TbsGbsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsAbsTb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsTbsTb
AbsTbsdTsdGsdCsdAsdAsdAsdAsdTsTbsCb
TbsTbsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
TbsGbsCbsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsAbsTb
CbsAbsTbsdTsdGsdCsdAsdAsdAsdAsTbsTb
AbsTbsTbsdGsdCsdAsdAsdAsdAsdTsTbsCb
TbsTbsGbsdCsdAsdAsdAsdAsdTsdTsCbsAb
TbsGbsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsAbsAbsTb
CbsAbsdTsdTsdGsdCsdAsdAsdAsAbsTbsTb
AbsTbsdTsdGsdCsdAsdAsdAsdAsTbsTbsCb
TbsTbsdGsdCsdAsdAsdAsdAsdTsTbsCbsAb
AbsdCsdAsdTsdTsdGsdCsdAsdAsAbsAb
CbsdAsdTsdTsdGsdCsdAsdAsdAsAbsTb
AbsdTsdTsdGsdCsdAsdAsdAsdAsTbsTb
TbsdTsdGsdCsdAsdAsdAsdAsdTsTbsCb
TbsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
GbsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsAb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsTb
AbsTbsdTsdGsdCsdAsdAsdAsdAsdTsTb
TbsTbsdGsdCsdAsdAsdAsdAsdTsdTsCb
TbsGbsdCsdAsdAsdAsdAsdTsdTsdCsAb
GbsCbsdAsdAsdAsdAsdTsdTsdCsdAsGb
AbsdCsdAsdTsdTsdGsdCsdAsdAsAb
CbsdAsdTsdTsdGsdCsdAsdAsdAsAb
AbsdTsdTsdGsdCsdAsdAsdAsdAsTb
TbsdTsdGsdCsdAsdAsdAsdAsdTsTb
TbsdGsdCsdAsdAsdAsdAsdTsdTsCb
GbsdCsdAsdAsdAsdAsdTsdTsdCsAb
CbsdAsdAsdAsdAsdTsdTsdCsdAsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsTbsTb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCb
AbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
TbsTbsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsTbsTb
CbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsTbsCb
AbsTbsTbsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
TbsTbsGbsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsAbsTbsTb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsTbsTbsCb
AbsTbsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAb
TbsTbsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
AbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCb
CbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
AbsTbsTbsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsTbsTbsCb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAb
AbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsTbsTbsCb
CbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAb
AbsTbsTbsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAb
CbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAb
CbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAb
CbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
AbsCbsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAbsGb
AbsCbsAbsTbsdTsdGsdCsdAsdAsdAsdAsdTsTbsCbsAbsGb
AbsCbsAbsdTsdTsdGsdCsdAsdAsdAsdAsTbsTbsCbsAbsGb
AbsCbsAbsTbsTbsdGsdCsdAsdAsdAsdAsdTsdTsCbsAbsGb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTbsGb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAb
TbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
TbsAbsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
GbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAb
AbsdGsdGsdTsdTsdAsdGsdGsdGsdC*sdTbsGb
GbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAb
TbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
TbsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sddTsGb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAb
TbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTb
TbsAbsdGsdGsdGsdC*sdTsdGsdAsdAsdTsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sTb
AbsdGsdGsdTsdTsdAsdGsdGsdGsdC*bsTb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsGb
GbsdGsdTsdTsdAsdGsdGsdGsdC*sTbsGb
GbsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAb
TbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAb
TbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAb
TbsAbsdGsdGsdGsdC*sdTsdGsdAsdAsTb
TbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
AbsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsdGsdGsdC*sdTsdGsdAsdAsdTsTb
AbsdGsdGsdTsdTsdAsdGsdGsdGsdC*b
GbsdGsdTsdTsdAsdGsdGsdGsdC*sTb
GbsdTsdTsdAsdGsdGsdGsdC*sdTsGb
TbsdTsdAsdGsdGsdGsdC*sdTsdGsAb
TbsdAsdGsdGsdGsdC*sdTsdGsdAsAb
AbsdGsdGsdGsdC*sdTsdGsdAsdAsTb
GbsdGsdGsdC*sdTsdGsdAsdAsdTsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
TbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAb
GbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAb
GbsTbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
TbsTbsAbsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsC*bsTbsGbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAbsAb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTb
TbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAbsAb
GbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTb
GbsTbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
GbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAbsAbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTb
GbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTb
GbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTb
GbsGbsTbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsdAsTbsTb
AbsGbsdGsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTbsTb
AbsGbsGbsTbsTbsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsGbsAbsAbsTbsTb
AbsGbsGbsdTsdTsdAsdGsdGsdGsdC*sdTsdGsAbsAbsTbsTb
AbsGbsGbsTbsdTsdAsdGsdGsdGsdC*sdTsdGsdAsAbsTbsTb
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsGbsC*b
AbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTb
AbsGbsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
C*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
AbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAb
TbsdAsdC*sdAsdAsdGsdC*sdAsdAsdGsGbsC*b
AbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTb
AbsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsC*b
AbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTb
AbsGbsdC*sdAsdAsdGsdGsdC*sdAsdTsdTsTb
TbsdAsdC*sdAsdAsdGsdC*sdAsdAsGbsGb
AbsdC*sdAsdAsdGsdC*sdAsdAsdGsGbsC*b
C*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAb
AbsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
AbsdGsdC*sdAsdAsdGsdGsdC*sdAsdTbsTb
GbsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsGb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*b
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sAb
AbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsTb
AbsGbsdC*sdAsdAsdGsdGsdC*sdAsddTsTb
GbsC*bsdAsdAsdGsdGsdC*sdAsdTsdTsTb
TbsdAsdC*sdAsdAsdGsdC*sdAsdAsGb
AbsdC*sdAsdAsdGsdC*sdAsdAsdGsGb
C*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*b
AbsdAsdGsdC*sdAsdAsdGsdGsdC*sAb
AbsdGsdC*sdAsdAsdGsdGsdC*sdAsdTb
GbsdC*sdAsdAsdGsdGsdC*sdAsdTsTb
C*bsdAsdAsdGsdGsdC*sdAsdTsdTsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTb
AbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAb
AbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
C*bsAbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTb
AbsAbsGbsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsGbsC*bsAb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAbsTb
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTb
AbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTb
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTb
AbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTb
C*bsAbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAbsTb
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTb
C*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsC*bsAbsTb
AbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTb
C*bsAbsAbsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbs
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbs
AbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTbs
AbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTbs
AbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsdTsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsdC*sdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTbsTb
TbsAbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTbsTb
TbsAbsC*bsAbsdAsdGsdC*sdAsdAsdGsdGsdC*sdAsTbsTbsTb
TbsAbsC*bsdAsdAsdGsdC*sdAsdAsdGsdGsdC*sAbsTbsTbsTb
TbsGbsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsAbsTb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTb
AbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
TbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
TbsGbsC*bsdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsAbsTb
C*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsTbsTb
AbsTbsTbsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
TbsTbsGbsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
TbsGbsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsAbsAbsTb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsAbsTbsTb
AbsTbsdTsdGsdC*sdAsdAsdAsdAsTbsTbsC*b
TbsTbsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAb
AbsdC*sdAsdTsdTsdGsdC*sdAsdAsAbsAb
C*bsdAsdTsdTsdGsdC*sdAsdAsdAsAbsTb
AbsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTb
TbsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
TbsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
GbsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsAb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsTb
AbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsTb
TbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsC*b
TbsGbsdC*sdAsdAsdAsdAsdTsdTsdC*sAb
GbsC*bsdAsdAsdAsdAsdTsdTsdC*sdAsGb
AbsdC*sdAsdTsdTsdGsdC*sdAsdAsAb
C*bsdAsdTsdTsdGsdC*sdAsdAsdAsAb
AbsdTsdTsdGsdC*sdAsdAsdAsdAsTb
TbsdTsdGsdC*sdAsdAsdAsdAsdTsTb
TbsdGsdC*sdAsdAsdAsdAsdTsdTsC*b
GbsdC*sdAsdAsdAsdAsdTsdTsdC*sAb
C*bsdAsdAsdAsdAsdTsdTsdC*sdAsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
AbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
TbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTb
C*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
AbsTbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
TbsTbsGbsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsAbsTbsTb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTbsC*b
AbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAb
TbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
AbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*b
C*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
AbsTbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTbsC*b
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAb
AbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTbsC*b
C*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAb
AbsTbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAb
C*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAb
C*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAb
C*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsdC*sAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
AbsC*bsdAsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAbsGb
AbsC*bsAbsTbsdTsdGsdC*sdAsdAsdAsdAsdTsTbsC*bsAbsGb
AbsC*bsAbsdTsdTsdGsdC*sdAsdAsdAsdAsTbsTbsC*bsAbsGb
AbsC*bsAbsTbsTbsdGsdC*sdAsdAsdAsdAsdTsdTsC*bsAbsGb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsGbAb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTbGb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsAbAb
TbTbdAsdGsdGsdGsdCsdTsdGsdAsAbTb
TbAbdGsdGsdGsdCsdTsdGsdAsdAsTbTb
GbdGsdTsdTsdAsdGsdGsdGsdCsdTsGbAb
AbdGsdGsdTsdTsdAsdGsdGsdGsdCsdTbGb
GbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAb
TbdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTb
TbdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsddTsGb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsAb
TbTbdAsdGsdGsdGsdCsdTsdGsdAsdAsTb
TbAbdGsdGsdGsdCsdTsdGsdAsdAsdTsTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsTb
AbdGsdGsdTsdTsdAsdGsdGsdGsdCbTb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsGb
GbdGsdTsdTsdAsdGsdGsdGsdCsTbGb
GbdTsdTsdAsdGsdGsdGsdCsdTsGbAb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsAb
TbTbdAsdGsdGsdGsdCsdTsdGsdAsAb
TbdTsdAsdGsdGsdGsdCsdTsdGsAbAb
TbAbdGsdGsdGsdCsdTsdGsdAsdAsTb
TbdAsdGsdGsdGsdCsdTsdGsdAsAbTb
AbdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbdGsdGsdCsdTsdGsdAsdAsdTsTb
AbdGsdGsdTsdTsdAsdGsdGsdGsdCb
GbdGsdTsdTsdAsdGsdGsdGsdCsTb
GbdTsdTsdAsdGsdGsdGsdCsdTsGb
TbdTsdAsdGsdGsdGsdCsdTsdGsAb
TbdAsdGsdGsdGsdCsdTsdGsdAsAb
AbdGsdGsdGsdCsdTsdGsdAsdAsTb
GbdGsdGsdCsdTsdGsdAsdAsdTsTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsGbAb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTb
TbTbdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsGbAb
GbGbTbdTsdAsdGsdGsdGsdCsdTsdGsAbAb
GbTbTbdAsdGsdGsdGsdCsdTsdGsdAsAbTb
TbTbAbdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsCbTbGbAb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsGbAbAb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsAbAbTb
TbTbdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsGbAbAb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAbTb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsGbAbAb
GbGbTbdTsdAsdGsdGsdGsdCsdTsdGsAbAbTb
GbTbTbdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsGbAbAb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAbTb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTb
GbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsGbAbAbTb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAbTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTb
GbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAbTb
GbGbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbGbTbdTsdAsdGsdGsdGsdCsdTsdGsAbAbTb
GbGbTbTbdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbGbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTbTb
AbGbdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAbTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbGbTbdTsdAsdGsdGsdGsdCsdTsdGsAbAbTbTb
AbGbGbTbTbdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsGbAbAbTbTb
AbGbGbdTsdTsdAsdGsdGsdGsdCsdTsdGsAbAbTbTb
AbGbGbTbdTsdAsdGsdGsdGsdCsdTsdGsdAsAbTbTb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsAbTb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsCbAb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsGbCb
AbAbdGsdCsdAsdAsdGsdGsdCsdAsTbTb
AbGbdCsdAsdAsdGsdGsdCsdAsdTsTbTb
CbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTb
AbdCsdAsdAsdGsdCsdAsdAsdGsdGsCbAb
TbdAsdCsdAsdAsdGsdCsdAsdAsdGsGbCb
AbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTb
AbdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsCb
AbAbdGsdCsdAsdAsdGsdGsdCsdAsdTsTb
AbGbdCsdAsdAsdGsdGsdCsdAsdTsdTsTb
TbdAsdCsdAsdAsdGsdCsdAsdAsGbGb
AbdCsdAsdAsdGsdCsdAsdAsdGsGbCb
CbdAsdAsdGsdCsdAsdAsdGsdGsCbAb
AbdAsdGsdCsdAsdAsdGsdGsdCsAbTb
AbdGsdCsdAsdAsdGsdGsdCsdAsdTbTb
GbdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsGb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsCb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsAb
AbAbdGsdCsdAsdAsdGsdGsdCsdAsTb
AbGbdCsdAsdAsdGsdGsdCsdAsddTsTb
GbCbdAsdAsdGsdGsdCsdAsdTsdTsTb
TbdAsdCsdAsdAsdGsdCsdAsdAsGb
AbdCsdAsdAsdGsdCsdAsdAsdGsGb
CbdAsdAsdGsdCsdAsdAsdGsdGsCb
AbdAsdGsdCsdAsdAsdGsdGsdCsAb
AbdGsdCsdAsdAsdGsdGsdCsdAsdTb
GbdCsdAsdAsdGsdGsdCsdAsdTsTb
CbdAsdAsdGsdGsdCsdAsdTsdTsTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsCbAb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTb
AbAbdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsCbAb
AbCbAbdAsdGsdCsdAsdAsdGsdGsdCsAbTb
CbAbAbdGsdCsdAsdAsdGsdGsdCsdAsTbTb
AbAbGbdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsGbCbAb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsCbAbTb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsAbTbTb
AbAbdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTb
AbCbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTb
CbAbAbdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsCbAbTb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTbTb
CbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsCbAbTb
AbCbAbdAsdGsdCsdAsdAsdGsdGsdCsAbTbTb
CbAbAbdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTb
AbCbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTbTb
AbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTbTb
AbCbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbCbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTbTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbCbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTbTbTb
TbAbCbAbdAsdGsdCsdAsdAsdGsdGsdCsAbTbTbTb
TbAbCbAbdAsdGsdCsdAsdAsdGsdGsdCsdAsTbTbTb
TbAbCbdAsdAsdGsdCsdAsdAsdGsdGsdCsAbTbTbTb
TbGbdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsAbTb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsTbTb
AbTbdTsdGsdCsdAsdAsdAsdAsdTsTbCb
TbTbdGsdCsdAsdAsdAsdAsdTsdTsCbAb
TbGbCbdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsAbTb
CbAbTbdTsdGsdCsdAsdAsdAsdAsTbTb
AbTbTbdGsdCsdAsdAsdAsdAsdTsTbCb
TbTbGbdCsdAsdAsdAsdAsdTsdTsCbAb
TbGbdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsAbAbTb
CbAbdTsdTsdGsdCsdAsdAsdAsAbTbTb
AbTbdTsdGsdCsdAsdAsdAsdAsTbTbCb
TbTbdGsdCsdAsdAsdAsdAsdTsTbCbAb
AbdCsdAsdTsdTsdGsdCsdAsdAsAbAb
CbdAsdTsdTsdGsdCsdAsdAsdAsAbTb
AbdTsdTsdGsdCsdAsdAsdAsdAsTbTb
TbdTsdGsdCsdAsdAsdAsdAsdTsTbCb
TbdGsdCsdAsdAsdAsdAsdTsdTsCbAb
GbdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsAb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsTb
AbTbdTsdGsdCsdAsdAsdAsdAsdTsTb
TbTbdGsdCsdAsdAsdAsdAsdTsdTsCb
TbGbdCsdAsdAsdAsdAsdTsdTsdCsAb
GbCbdAsdAsdAsdAsdTsdTsdCsdAsGb
AbdCsdAsdTsdTsdGsdCsdAsdAsAb
CbdAsdTsdTsdGsdCsdAsdAsdAsAb
AbdTsdTsdGsdCsdAsdAsdAsdAsTb
TbdTsdGsdCsdAsdAsdAsdAsdTsTb
TbdGsdCsdAsdAsdAsdAsdTsdTsCb
GbdCsdAsdAsdAsdAsdTsdTsdCsAb
CbdAsdAsdAsdAsdTsdTsdCsdAsGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsTbTb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCb
AbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAb
TbTbdGsdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsTbTb
CbAbTbdTsdGsdCsdAsdAsdAsdAsdTsTbCb
AbTbTbdGsdCsdAsdAsdAsdAsdTsdTsCbAb
TbTbGbdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsAbTbTb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsTbTbCb
AbTbdTsdGsdCsdAsdAsdAsdAsdTsTbCbAb
TbTbdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAb
AbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCb
CbAbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAb
AbTbTbdGsdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsTbTbCb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCbAb
AbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsTbTbCb
CbAbTbdTsdGsdCsdAsdAsdAsdAsdTsTbCbAb
AbTbTbdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAb
CbAbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCbAb
CbAbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCbAb
CbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCs
AbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCs
AbGb
AbCbAbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbAbTbdTsdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsCb
AbGb
AbCbdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCbAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsdTsTbCbAbGb
AbCbAbTbdTsdGsdCsdAsdAsdAsdAsdTsTbCbAbGb
AbCbAbdTsdTsdGsdCsdAsdAsdAsdAsTbTbCbAbGb
AbCbAbTbTbdGsdCsdAsdAsdAsdAsdTsdTsCbAbGb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssGbssAb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTbssGb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssAbssAb
TbssTbssdAssdGssdGssdGssdCssdTssdGssdAssAbssTb
TbssAbssdGssdGssdGssdCssdTssdGssdAssdAssTbssTb
GbssdGssdTssdTssdAssdGssdGssdGssdCssdTssGbssAb
AbssdGssdGssdTssdTssdAssdGssdGssdGssdCssdTbssGb
GbssdTssdTssdAssdGssdGssdGssdCssdTssdGssAbssAb
TbssdTssdAssdGssdGssdGssdCssdTssdGssdAssAbssTb
TbssdAssdGssdGssdGssdCssdTssdGssdAssdAssTbssTb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssddTssGb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb
TbssTbssdAssdGssdGssdGssdCssdTssdGssdAssdAssTb
TbssAbssdGssdGssdGssdCssdTssdGssdAssdAssdTssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssTb
AbssdGssdGssdTssdTssdAssdGssdGssdGssdCbssTb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssGb
GbssdGssdTssdTssdAssdGssdGssdGssdCssTbssGb
GbssdTssdTssdAssdGssdGssdGssdCssdTssGbssAb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssAb
TbssTbssdAssdGssdGssdGssdCssdTssdGssdAssAb
TbssdTssdAssdGssdGssdGssdCssdTssdGssAbssAb
TbssAbssdGssdGssdGssdCssdTssdGssdAssdAssTb
TbssdAssdGssdGssdGssdCssdTssdGssdAssAbssTb
AbssdGssdGssdGssdCssdTssdGssdAssdAssTbssTb
AbssGbssdGssdGssdCssdTssdGssdAssdAssdTssTb
AbssdGssdGssdTssdTssdAssdGssdGssdGssdCb
GbssdGssdTssdTssdAssdGssdGssdGssdCssTb
GbssdTssdTssdAssdGssdGssdGssdCssdTssGb
TbssdTssdAssdGssdGssdGssdCssdTssdGssAb
TbssdAssdGssdGssdGssdCssdTssdGssdAssAb
AbssdGssdGssdGssdCssdTssdGssdAssdAssTb
GbssdGssdGssdCssdTssdGssdAssdAssdTssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssGbssAb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssAbssAb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssdAssAbssTb
TbssTbssdAssdGssdGssdGssdCssdTssdGssdAssdAssTbssTb
AbssGbssGbssdTssdTssdAssdGssdGssdGssdCssdTssGbssAb
GbssGbssTbssdTssdAssdGssdGssdGssdCssdTssdGssAbssAb
GbssTbssTbssdAssdGssdGssdGssdCssdTssdGssdAssAbssTb
TbssTbssAbssdGssdGssdGssdCssdTssdGssdAssdAssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssCbssTbssGbssAb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssGbssAbssAb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssAbssAbssTb
TbssTbssdAssdGssdGssdGssdCssdTssdGssdAssAbssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssGbssAbssAb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssAbssAbssTb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssdAssAbssTbssTb
AbssGbssGbssdTssdTssdAssdGssdGssdGssdCssdTssGbssAbssAb
GbssGbssTbssdTssdAssdGssdGssdGssdCssdTssdGssAbssAbssTb
GbssTbssTbssdAssdGssdGssdGssdCssdTssdGssdAssAbssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssGbssAbssAb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssAbssAbssTb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssdAssAbssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssAbssAb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAssAbssTb
GbssTbssdTssdAssdGssdGssdGssdCssdTssdGssdAssdAssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssGbss
AbssAbssTb
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssAbss
AbssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGss
GbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
AbssGbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGss
AbssAbssTb
GbssGbssTbssdTssdAssdGssdGssdGssdCssdTssdGssdAss
AbssTbssTb
AbssGbssGbssTbssdTssdAssdGssdGssdGssdCssdTssdGss
AbssAbssTb
GbssGbssTbssTbssdAssdGssdGssdGssdCssdTssdGssdAss
AbssTbssTb
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGss
AbssGbssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGss
AbssGbssGbssdTssdTssdAssdGssdGssdGssdCssdTssdGss
AbssAbssTbssTb
AbssAbssTbssTb
AbssAbssTbssTb
AbssAbssTbssTb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssCbssAb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssGbssCb
AbssAbssdGssdCssdAssdAssdGssdGssdCssdAssTbssTb
AbssGbssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
CbssdAssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
AbssdCssdAssdAssdGssdCssdAssdAssdGssdGssCbssAb
TbssdAssdCssdAssdAssdGssdCssdAssdAssdGssGbssCb
AbssdAssdGssdCssdAssdAssdGssdGssdCssdAssTbssTb
AbssdGssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAssTb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssCb
AbssAbssdGssdCssdAssdAssdGssdGssdCssdAssdTssTb
AbssGbssdCssdAssdAssdGssdGssdCssdAssdTssdTssTb
TbssdAssdCssdAssdAssdGssdCssdAssdAssGbssGb
AbssdCssdAssdAssdGssdCssdAssdAssdGssGbssCb
CbssdAssdAssdGssdCssdAssdAssdGssdGssCbssAb
AbssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
AbssdGssdCssdAssdAssdGssdGssdCssdAssdTbssTb
GbssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssGb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssCb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssAb
AbssAbssdGssdCssdAssdAssdGssdGssdCssdAssTb
AbssGbssdCssdAssdAssdGssdGssdCssdAssddTssTb
GbssCbssdAssdAssdGssdGssdCssdAssdTssdTssTb
TbssdAssdCssdAssdAssdGssdCssdAssdAssGb
AbssdCssdAssdAssdGssdCssdAssdAssdGssGb
CbssdAssdAssdGssdCssdAssdAssdGssdGssCb
AbssdAssdGssdCssdAssdAssdGssdGssdCssAb
AbssdGssdCssdAssdAssdGssdGssdCssdAssdTb
GbssdCssdAssdAssdGssdGssdCssdAssdTssTb
CbssdAssdAssdGssdGssdCssdAssdTssdTssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssCbssAb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAssTbssTb
AbssAbssdGssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssCbssAb
AbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
CbssAbssAbssdGssdCssdAssdAssdGssdGssdCssdAssTbssTb
AbssAbssGbssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssGbssCbssAb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssCbssAbssTb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssAbssTbssTb
AbssAbssdGssdCssdAssdAssdGssdGssdCssdAssTbssTbssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAssTbssTb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssAbssTb
AbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAssTbssTb
CbssAbssAbssdGssdCssdAssdAssdGssdGssdCssdAssdTssTbssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssCbssAbssTb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssAbssTbssTb
CbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAssTbssTbssTb
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssCbssAbssTb
AbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCssAbssTbssTb
CbssAbssAbssdGssdCssdAssdAssdGssdGssdCssdAssTbssTbssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAss
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAss
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssTbssTb
AbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAss
TbssTbssTb
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssTbssTb
AbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCssdAss
TbssTbssTb
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssTbssTbssTb
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssTbssTbssTb
TbssAbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCss
AbssTbssTbssTb
TbssAbssCbssAbssdAssdGssdCssdAssdAssdGssdGssdCss
TbssAbssCbssdAssdAssdGssdCssdAssdAssdGssdGssdCss
AbssTbssTbssTb
TbssGbssdCssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssAbssTb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssTbssTb
AbssTbssdTssdGssdCssdAssdAssdAssdAssdTssTbssCb
TbssTbssdGssdCssdAssdAssdAssdAssdTssdTssCbssAb
TbssGbssCbssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssAbssTb
CbssAbssTbssdTssdGssdCssdAssdAssdAssdAssTbssTb
AbssTbssTbssdGssdCssdAssdAssdAssdAssdTssTbssCb
TbssTbssGbssdCssdAssdAssdAssdAssdTssdTssCbssAb
TbssGbssdCssdAssdAssdAssdAssdTssdTssCbssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssAbssAbssTb
CbssAbssdTssdTssdGssdCssdAssdAssdAssAbssTbssTb
AbssTbssdTssdGssdCssdAssdAssdAssdAssTbssTbssCb
TbssTbssdGssdCssdAssdAssdAssdAssdTssTbssCbssAb
AbssdCssdAssdTssdTssdGssdCssdAssdAssAbssAb
CbssdAssdTssdTssdGssdCssdAssdAssdAssAbssTb
AbssdTssdTssdGssdCssdAssdAssdAssdAssTbssTb
TbssdTssdGssdCssdAssdAssdAssdAssdTssTbssCb
TbssdGssdCssdAssdAssdAssdAssdTssdTssCbssAb
GbssdCssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssAb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssTb
AbssTbssdTssdGssdCssdAssdAssdAssdAssdTssTb
TbssTbssdGssdCssdAssdAssdAssdAssdTssdTssCb
TbssGbssdCssdAssdAssdAssdAssdTssdTssdCssAb
GbssCbssdAssdAssdAssdAssdTssdTssdCssdAssGb
AbssdCssdAssdTssdTssdGssdCssdAssdAssAb
CbssdAssdTssdTssdGssdCssdAssdAssdAssAb
AbssdTssdTssdGssdCssdAssdAssdAssdAssTb
TbssdTssdGssdCssdAssdAssdAssdAssdTssTb
TbssdGssdCssdAssdAssdAssdAssdTssdTssCb
GbssdCssdAssdAssdAssdAssdTssdTssdCssAb
CbssdAssdAssdAssdAssdTssdTssdCssdAssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssTbssTb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssTbssCb
AbssTbssdTssdGssdCssdAssdAssdAssdAssdTssdTssCbssAb
TbssTbssdGssdCssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssTbssTb
CbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTssTbssCb
AbssTbssTbssdGssdCssdAssdAssdAssdAssdTssdTssCbssAb
TbssTbssGbssdCssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssAbssTbssTb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssTbssTbssCb
AbssTbssdTssdGssdCssdAssdAssdAssdAssdTssTbssCbssAb
TbssTbssdGssdCssdAssdAssdAssdAssdTssdTssCbssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssTbssCb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssCbssAb
AbssTbssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssTbssCb
CbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTssdTssCbssAb
AbssTbssTbssdGssdCssdAssdAssdAssdAssdTssdTssdCssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssTbssTbssCb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssTbssCbssAb
AbssTbssdTssdGssdCssdAssdAssdAssdAssdTssdTssCbssAbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssTbssTbssCb
CbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTssTbssCbssAb
AbssTbssTbssdGssdCssdAssdAssdAssdAssdTssdTssCbssAbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTss
CbssAb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
AbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTss
CbssAb
CbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
AbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssTbss
CbssAb
CbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTssdTssCbss
AbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssTbss
CbssAb
CbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssCbss
AbssGb
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTss
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTss
AbssCbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTss
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTss
AbssCbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTss
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTss
AbssCbssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTss
TbssCbssAbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssdTss
TbssCbssAbssGb
AbssCbssAbssTbssdTssdGssdCssdAssdAssdAssdAssdTss
TbssCbssAbssGb
AbssCbssAbssdTssdTssdGssdCssdAssdAssdAssdAssTbss
TbssCbssAbssGb
AbssCbssAbssTbssTbssdGssdCssdAssdAssdAssdAssdTss
The following antisense-oligonucleotides in form of gapmers as listed in Table 30 to Table 32 are especially preferred.
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTb1sGb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1s
Tb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1s
Gb
2
sdGsdTsdTsdAsdGsdGsdGsdCsdTsGb2sAb2
Ab
2
sdGsdGsdTsdTsdAsdGsdGsdGsdCsdTb2sGb2
Gb
2
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb2sAb2
Tb
2
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb2sTb1
Gb
3
sGb
3
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb3
Ab
3
sGb
3
sdGsdTsdTsdAsdGsdGsdGsdCsddTsGb3
Gb
3
sTb
3
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb3
Tb
3
sTb
3
sdAsdGsdGsdGsdCsdTsdGsdAsdAsTb3
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTb1Gb1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1
Gb
4dGsdTsdTsdAsdGsdGsdGsdCsdTsGb4Ab4
Ab
4dGsdGsdTsdTsdAsdGsdGsdGsdCsdTb4Gb4
Gb
4dTsdTsdAsdGsdGsdGsdCsdTsdGsAb4Ab4
Tb
4dTsdAsdGsdGsdGsdCsdTsdGsdAsAb4Tb4
Gb
5
Gb
5dTsdTsdAsdGsdGsdGsdCsdTsdGsAb5
Ab
5
Gb
5dGsdTsdTsdAsdGsdGsdGsdCsddTsGb5
Gb
5
Tb
5dTsdAsdGsdGsdGsdCsdTsdGsdAsAb5
Tb
5
Tb
5dAsdGsdGsdGsdCsdTsdGsdAsdAsTb5
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTb1ssGb1
Gb
1
ssTb1ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ssAb1
Tb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssAb1ssTb1
Gb
6
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssGb6ssAb6
Ab
6
ssdGssdGssdTssdTssdAssdGssdGssdGssdCssdTb6ssGb6
Gb
6
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb6ssAb6
Tb
6
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb6ssTb6
Gb
7
ssGb
7
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb7
Ab
7
ssGb
7
ssdGssdTssdTssdAssdGssdGssdGssdCssddTssGb7
Gb
7
ssTb
7
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb7
Tb
7
ssTb
7
ssdAssdGssdGssdGssdCssdTssdGssdAssdAssTb7
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsdAsTb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1
Gb
1
sdGsdTsdTsdAsdGsdGsdGsdCsTb1sGb1
Gb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1
Tb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1
Tb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsTb1Gb1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1
Gb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssTb1ssGb1
Gb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1
Gb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1
Tb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssAb1
Tb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ssAb1
Gb
1
sdGsdTsdTsdAsdGsdGsdGsdC*sTb1
Gb
1
sdTsdTsdAsdGsdGsdGsdC*sdTsGb1
Tb
1
sdTsdAsdGsdGsdGsdC*sdTsdGsAb1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsTb1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1
Gb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssTb1
Gb
1
ssdTssdTssdAssdGssdGssdGssdC*ssdTssGb1
Tb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1
Tb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1
Gb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1
Gb
1
sTb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1
Tb
1
sTb
1
sAb
1
sdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sdG
1
sdTsdTsdAsdGsdGsdGsC*b1sTb1sGb1sAb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1
Tb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1
Gb
1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1
Tb
1
Tb
1
Ab
1dGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsCb1Tb1Gb1Ab1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ssAb1
Gb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb1ssTb1
Tb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssdAssTb1ssTb1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1
Gb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ssAb1
Gb
1
ssTb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssAb1ssTb1
Tb
1
ssTb
1
ssAb
1
ssdGssdGssdGssdCssdTssdGssdAssdAssTb1ssTb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssCb1ssTb1ssGb1ssAb1
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1ssAb1
Gb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ssAb1ssTb1
Tb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssAbssTbssTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1
Gb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1
Gb
1
sTb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1
Gb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1
Gb
1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssGb1ss
Ab
1
ssAb
1
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
Gb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb1ss
Tb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ss
Ab
1
ssAb
1
Gb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
Gb
1
ssTb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssAb1ss
Tb
1
ssTb
1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssGb1ss
Ab
1
ssAb
1
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
Gb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb1ss
Tb
1
ssTb
1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssd
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssd
Gb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssd
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1sTb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1
Gb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1
Gb
1
sGb
1
sTb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1Tb1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb1Gb1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1
Gb
1Tb1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1
Gb
1
Gb
1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1ss
Ab
1
ssTb
1
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ssAb1ss
Tb
1
ssTb
1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGss
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
Gb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAssAb1ss
Tb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
Gb
1
ssGb
1
ssTb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAssAb1ss
Tb
1
ssTb
1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sTb1sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
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1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1
ssTb
1
ssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1
ssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
C*b
1
sAb
1
sdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1
Ab
1
sC*bsdAsdAsdGsdCsdAsdAsdGsdGsC*b1sAb1
Tb
1
sAb
1
sdCsdAsdAsdGsdCsdAsdAsdGsGb1sC*b1
Ab
1
sAb
1
sdGsdCsdAsdAsdGsdGsdCsdAsTb1sTb1
Ab
1
sGb
1
sdCsdAsdAsdGsdGsdCsdAsdTsTb1sTb1
C*b
2
sdAsdAsdGsdCsdAsdAsdGsdGsdCsAb2sTb2
Ab
2
sdCsdAsdAsdGsdCsdAsdAsdGsdGsC*b2sAb2
Tb
2
sdAsdCsdAsdAsdGsdCsdAsdAsdGsGb2sC*b2
Ab
2
sdAsdGsdCsdAsdAsdGsdGsdCsdAsTb2sTb2
Ab
2
sdGsdCsdAsdAsdGsdGsdCsdAsdTsTb2sTb2
C*b
3
sAb
3
sdAsdGsdCsdAsdAsdGsdGsdCsdAsTb3
Ab
3
sC*b
3
sdAsdAsdGsdCsdAsdAsdGsdGsdCsAb3
Tb
3
sAb
3
sdCsdAsdAsdGsdCsdAsdAsdGsdGsC*b3
Ab
3
sAb
3
sdGsdCsdAsdAsdGsdGsdCsdAsdTsTb3
Ab
3
sGb
3
sdCsdAsdAsdGsdGsdCsdAsdTsdTsTb3
C*b
1
Ab
1dAsdGsdCsdAsdAsdGsdGsdCsAb1Tb1
Ab
1
Cb
1dAsdAsdGsdCsdAsdAsdGsdGsC*b1Ab1
Tb
1
Ab
1dCsdAsdAsdGsdCsdAsdAsdGsGb1C*b1
Ab
1
Ab
1dGsdCsdAsdAsdGsdGsdCsdAsTb1Tb1
Ab
1
Gb
1dCsdAsdAsdGsdGsdCsdAsdTsTb1Tb1
C*b
4dAsdAsdGsdCsdAsdAsdGsdGsdCsAb4Tb4
Ab
4dCsdAsdAsdGsdCsdAsdAsdGsdGsC*b4Ab4
Tb
4dAsdCsdAsdAsdGsdCsdAsdAsdGsGb4C*b4
Ab
4dAsdGsdCsdAsdAsdGsdGsdCsdAsTb4Tb4
Ab
4dGsdCsdAsdAsdGsdGsdCsdAsdTsTb4Tb4
C*b
5
Ab
5dAsdGsdCsdAsdAsdGsdGsdCsdAsTb5
Ab
5
C*b
5dAsdAsdGsdCsdAsdAsdGsdGsdCsAb5
Tb
5
Ab
5dCsdAsdAsdGsdCsdAsdAsdGsdGsC*b5
Ab
5
Ab
5dGsdCsdAsdAsdGsdGsdCsdAsdTsTb5
Ab
5
Gb
5dCsdAsdAsdGsdGsdCsdAsdTsdTsTb5
Ab
1
ssCb
1
ssdAssdAssdGssdCssdAssdAssdGssdGssC*b1ssAb1
Tb
1
ssAb
1
ssdCssdAssdAssdGssdCssdAssdAssdGssGb1ssC*b1
Ab
1
ssAb
1
ssdGssdCssdAssdAssdGssdGssdCssdAssTb1ssTb1
Ab
1
ssGb
1
ssdCssdAssdAssdGssdGssdCssdAssdTssTb1ssTb1
C*b
6
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb6ssTb6
Ab
6
ssdCssdAssdAssdGssdCssdAssdAssdGssdGssC*b6ssAb6
Tb
6
ssdAssdCssdAssdAssdGssdCssdAssdAssdGssGb6ssC*b6
Ab
6
ssdAssdGssdCssdAssdAssdGssdGssdCssdAssTb6ssTb6
Ab
6
ssdGssdCssdAssdAssdGssdGssdCssdAssdTssTb6ssTb6
C*b
7
ssAb
7
ssdAssdGssdCssdAssdAssdGssdGssdCssdAssTb7
Ab
7
ssC*b
7
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb7
Tb
7
ssAb
7
ssdCssdAssdAssdGssdCssdAssdAssdGssdGssC*b7
Ab
7
ssAb
7
ssdGssdCssdAssdAssdGssdGssdCssdAssdTssTb7
Ab
7
ssGb
7
ssdCssdAssdAssdGssdGssdCssdAssdTssdTssTb7
Ab
1
sdCsdAsdAsdGsdCsdAsdAsdGsGb1sC*b1
C*b
1
sdAsdAsdGsdCsdAsdAsdGsdGsC*b1sAb1
Ab
1
sdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1
Tb
1
ssAb
1
ssC*b
1
ssAb
1
ssdAssdGssdCssdAssdAssdGssdGssdCssdAssTb1ss
Tb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb1ss
Tb
1
ssTb
1
ssTb
1
Tb
1
sGb
1
sdCsdAsdAsdAsdAsdTsdTsdCsAb1sGb1
Ab
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1
Tb
1
sTb
1
sdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1
Tb
2
sGb
2
sC*bsdAsdAsdAsdAsdTsdTsdC*sAb2sGb2
Ab
2
sTb
2
sTb
2
sdGsdCsdAsdAsdAsdAsdTsTb2sC*b2
Tb
2
sTb
2
sGb
2
sdCsdAsdAsdAsdAsdTsdTsC*b2sAb2
Tb
3
sGb
3
sdCsdAsdAsdAsdAsdTsdTsC*b3sAb3sGb3
Ab
3
sTb
3
sdTsdGsdCsdAsdAsdAsdAsTb3sTb3sC*b3
Tb
3
sTb
3
sdGsdCsdAsdAsdAsdAsdTsTb3sC*b3sAb3
Tb
1
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1dCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
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1dTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1
Tb
1
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1dGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1
Tb
4
Gb
4
C*b
4dAsdAsdAsdAsdTsdTsdCsAb4Gb4
Ab
4
Tb
4
Tb
4dGsdCsdAsdAsdAsdAsdTsTb4C*b4
Tb
4
Tb
4
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4dCsdAsdAsdAsdAsdTsdTsC*b4Ab4
Tb
5
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5dCsdAsdAsdAsdAsdTsdTsC*b5Ab5Gb5
Ab
5
Tb
5dTsdGsdCsdAsdAsdAsdAsTb5Tb5C*b5
Tb
5
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5dGsdCsdAsdAsdAsdAsdTsTb5C*b5Ab5
Tb
1
ssGb
1
ssdCssdAssdAssdAssdAssdTssdTssdCssAb1ssGb1
Ab
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssTb1ssCb1
Tb
1
ssTb
1
ssdGssdCssdAssdAssdAssdAssdTssdTssC*b1ssAb1
Tb
6
ssGb
6
ssC*b
6
ssdAssdAssdAssdAssdTssdTssdCssAb6ssGb6
Ab
6
ssTb
6
ssTb
6
ssdGssdCssdAssdAssdAssdAssdTssTb6ssC*b6
Tb
6
ssTb
6
ssGb
6
ssdCssdAssdAssdAssdAssdTssdTssC*b6ssAb6
Tb
7
ssGb
7
ssdCssdAssdAssdAssdAssdTssdTssC*b7ssAb7ssGb7
Ab
7
ssTb
7
ssdTssdGssdCssdAssdAssdAssdAssTb7ssTb7ssC*b7
Tb
7
ssTb
7
ssdGssdCssdAssdAssdAssdAssdTssTb7ssC*b7ssAb7
Tb
1
sdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1
Tb
1
sdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1
Gb
1
sdCsdAsdAsdAsdAsdTsdTsdCsAb1sGb1
Tb
1
sTb
1
sdGsdCsdAsdAsdAsdAsdTsdTsC*b1
Tb
1
sGb
1
sdCsdAsdAsdAsdAsdTsdTsdCsAb1
Gb
1
sC*b
1
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Tb
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Tb
1dGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1
Gb
1dCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Tb
1
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1dGsdCsdAsdAsdAsdAsdTsdTsC*b1
Tb
1
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1dCsdAsdAsdAsdAsdTsdTsdCsAb1
Gb
1
C*b
1dAsdAsdAsdAsdTsdTsdCsdAsGb1
Tb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssTb1ssC*b1
Tb
1
ssdGssdCssdAssdAssdAssdAssdTssdTssC*b1ssAb1
Gb
1
ssdCssdAssdAssdAssdAssdTssdTssdCssAb1ssGb1
Tb
1
ssTb
1
ssdGssdCssdAssdAssdAssdAssdTssdTssC*b1
Tb
1
ssGb
1
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Gb
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1
ssdAssdAssdAssdAssdTssdTssdCssdAssGb1
Tb
1
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Gb
1
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C*b
1
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Tb
1dGsdCsdAsdAsdAsdAsdTsdTsC*b1
Gb
1dCsdAsdAsdAsdAsdTsdTsdCsAb1
C*b
1dAsdAsdAsdAsdTsdTsdCsdAsGb1
Tb
1
ssdGssdCssdAssdAssdAssdAssdTssdTssC*b1
Gb
1
ssdCssdAssdAssdAssdAssdTssdTssdCssAb1
C*b
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C*b
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1
sdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1
Ab
1
sTb
1
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Tb
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1
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C*b
1
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1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1
Ab
1
sTb
1
sTb
1
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Tb
1
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1
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1
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C*b
1
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1
sdTsdTsdGsdCsdAsdAsdAsdAsTb1sTb1sC*b1
Ab
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1
Tb
1
sTb
1
sdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
C*b
1
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1dTsdTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1
Ab
1
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1dTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1
Tb
1
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1dGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1
Ab
1
Tb
1
Tb
1dGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1
Tb
1
Tb
1
Gb
1dCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
C*b
1
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1dTsdTsdGsdCsdAsdAsdAsdAsTb1Tb1C*b1
Ab
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Tb
1dTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1
Tb
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1dGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
C*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssdTssTb1ssC*b1
Ab
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssdTssC*b1ssAb1
Tb
1
ssTb
1
ssdGssdCssdAssdAssdAssdAssdTssdTssdCssAb1ssGb1
C*b
1
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1
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1
ssdTssdGssdCssdAssdAssdAssdAssdTssTb1ssC*b1
Ab
1
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1
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1
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Tb
1
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1
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1
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C*b
1
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1
ssdTssdTssdGssdCssdAssdAssdAssdAssTb1ssTb1ssC*b1
Ab
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssTb1ssC*b1ssAb1
Tb
1
ssTb
1
ssdGssdCssdAssdAssdAssdAssdTssdTssC*b1ssAb1ssGb1
Ab
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1
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C*b
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1
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Ab
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1
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Ab
1
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1
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1
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C*b
1
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1
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1
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Ab
1
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1
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1
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Ab
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1
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C*b
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Ab
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
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1
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1
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C*b
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1
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1
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Ab
1
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1
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Ab
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C*b
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C*b
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1dTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
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C*b
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1
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1dTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1
Ab
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Ab
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C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsTb1Tb1C*b1
C*b
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1dTsdTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1
Ab
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C*b
1
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C*b
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C*b
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C*b
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C*b
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C*b
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C*b
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C*b
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C*b
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C*b
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sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
sC*b
1
sdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
sC*b
1
sdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsTb1sTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sTb
1
sdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsTb1Tb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1
Tb
1
Tb
1dGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
ssC*b
1
ssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
Ab
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
Ab
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
Ab
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssTb1ss
Ab
1
ssC*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssdTssTb1ss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssTb1ss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssTb1ssTb1ss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssTb
1
ssTbssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Also especially referred are the a mer antisense-oligonucleotides of Table 33.
Gb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1
Gb
3
sGb
3
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb3
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1
Gb
4dGsdTsdTsdAsdGsdGsdGsdCsdTsGb4Ab4
Gb
5
Gb
5dTsdTsdAsdGsdGsdGsdCsdTsdGsAb5
Gb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1
Gb
6
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssGb6ssAb6
Gb
7
ssGb
7
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb7
Ab
1
sGb
1sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1sTb1
Ab
1
sGb
1
sdGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sTb
1
sdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsGb1sAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1sAb1sTb1sTb1
Ab
1
sGb
1
sGb
1
sTb
1
sdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1sTb1sTb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsdAsTb1Tb1
Ab
1
Gb
1dGsdTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1
Tb
1dAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsGb1Ab1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1dTsdTsdAsdGsdGsdGsdCsdTsdGsAb1Ab1Tb1Tb1
Ab
1
Gb
1
Gb
1
Tb
1dTsdAsdGsdGsdGsdCsdTsdGsdAsAb1Tb1Tb1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1
ssGb
1ssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1
ssGb
1
ssTb
1ssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssGb
1ssdGssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb ssdTssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1ssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1
ssTb
1ssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssdTssdTssdAssdGssdGssdGssdCssdTssdGssAb1ss
Ab
1
ssTb
1
ssTb
1
Ab
1
ssGb
1
ssGb
1
ssTb
1ssdTssdAssdGssdGssdGssdCssdTssdGssdAss
Ab
1
ssTb
1
ssTb
1
C*b
1
sAb
1sdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1
C*b
2sdAsdAsdGsdCsdAsdAsdGsdGsdCsAb2sTb2
C*b
3
sAb
3
sdAsdGsdCsdAsdAsdGsdGsdCsdAsTb3
C*b
1
Ab
1dAsdGsdCsdAsdAsdGsdGsdCsAb1Tb1
C*b
4dAsdAsdGsdCsdAsdAsdGsdGsdCsAb4Tb4
C*b
1
ssAb
1
ssdAssdGssdCssdAssdAssdGssdGssdCssAb1ssTb1
C*b
6ssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb6ssTb6
C*b
7
ssAb
7
ssdAssdGssdCssdAssdAssdGssdGssdCssdAssTb7
Tb
1
sAb
1
sdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb1sTb1
Tb
1
sAb
1
sC*bsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb1sTb1
Tb
1
sAb
1
sC*b
1
sAb
1
sdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb1sTb1
Tb
1
sAb
1
sdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTb1sTb1sTb1
Tb
1
sAb
1
sdCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1sTb1sTb1
Tb
1
sAb
1
sC*bsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTb1sTb1sTb1
Tb
1
sAb
1
sC*b
1
sAb
1
sdAsdGsdCsdAsdAsdGsdGsdCsdAsTb1sTb1sTb1
Tb
1
sAb
1
sC*bsdAsdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1sTb1sTb1
Tb
1
sAb
1
sC*b
1
sAb
1
sdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1sTb1sTb1
Tb
1
sAb
1
sC*b
1
sAb
1
sdAsdGsdCsdAsdAsdGsdGsdCsdAsTb1sTb1sTb1
Tb
1
sAb
1
sC*bsdAsdAsdGsdCsdAsdAsdGsdGsdCsAb1sTb1sTb1sTb1
Tb
1
Ab
1dCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb1Tb1
Tb
1
Ab
1
C*b
1dAsdAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb1Tb1
Tb
1
Ab
1
C*b
1
Ab
1dAsdGsdCsdAsdAsdGsdGsdCsdAsdTsTb1Tb1
Tb
1
Ab
1dCsdAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTb1Tb1Tb1
Tb
1
Ab
1dCsdAsdAsdGsdCsdAsdAsdGsdGsdCsAb1Tb1Tb1Tb1
Tb
1
Ab
1
C*b
1dAsdAsdGsdCsdAsdAsdGsdGsdCsdAsTb1Tb1Tb1
Tb
1
Ab
1
C*b
1
Ab
1dAsdGsdCsdAsdAsdGsdGsdCsdAsTb1Tb1Tb1
Tb
1
Ab
1
C*b
1dAsdAsdGsdCsdAsdAsdGsdGsdCsAb1Tb1Tb1Tb1
Tb
1
Ab
1
C*b
1
Ab
1dAsdGsdCsdAsdAsdGsdGsdCsAb1Tb1Tb1Tb1
Tb
1
Ab
1
C*b
1
Ab
1dAsdGsdCsdAsdAsdGsdGsdCsdAsTb1Tb1Tb1
Tb
1
Ab
1
C*b
1dAsdAsdGsdCsdAsdAsdGsdGsdCsAb1Tb1Tb1Tb1
Tb
1
ssAb
1
ssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAssdTss
Tb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAssdTss
Tb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssAb
1
ssdAssdGssdCssdAssdAssdGssdGssdCssdAss
Tb
1
ssAb
1
ssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAssTb1ss
Tb
1
ssTb
1
Tb
1
ssAb
1
ssdCssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb1ssTb1ss
Tb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssdAss
Tb
1
ssTb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssAb
1
ssdAssdGssdCssdAssdAssdGssdGssdCssdAss
Tb
1
ssTb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb1ss
Tb
1
ssTb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssAb
1
ssdAssdGssdCssdAssdAssdGssdGssdCssAb1ss
Tb
1
ssTb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssAb
1
ssdAssdGssdCssdAssdAssdGssdGssdCssdAss
Tb
1
ssTb
1
ssTb
1
Tb
1
ssAb
1
ssC*b
1
ssdAssdAssdGssdCssdAssdAssdGssdGssdCssAb1ss
Tb
1
ssTb
1
ssTb
1
Tb
1
sGb
1
sdCsdAsdAsdAsdAsdTsdTsdCsAb1sGb1
Tb
2
sGb
2
sC*b
2sdAsdAsdAsdAsdTsdTsdC*sAb2sGb2
Tb
3
sGb
3
sdCsdAsdAsdAsdAsdTsdTsC*b3sAb3sGb3
Tb
1
Gb
1dCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Tb
4
Gb
4
C*b
4dAsdAsdAsdAsdTsdTsdCsAb4Gb4
Tb
5
Gb
5dCsdAsdAsdAsdAsdTsdTsC*b5Ab5Gb5
Tb
1
ssGb
1
ssdCssdAssdAssdAssdAssdTssdTssdCssAb1ssGb1
Tb
6
ssGbossC*b6ssdAssdAssdAssdAssdTssdTssdCssAb6ssGb6
Tb
7
ssGb
7
ssdCssdAssdAssdAssdAssdTssdTssC*b7ssAb7ssGb7
Ab
1
sC*bsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1sGb1
Ab
1
sC*b
1
sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1sGb1
Ab
1
sC*b
1
sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
sC*bsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
sC*bsdAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sdTsdGsdCsdAsdAsdAsdAsdTsTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sdTsdTsdGsdCsdAsdAsdAsdAsTb1sTb1sC*b1sAb1sGb1
Ab
1
sC*b
1
sAb
1
sTb
1
sTb
1
sdGsdCsdAsdAsdAsdAsdTsdTsC*b1sAb1sGb1
Ab
1
C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsdTsdCsAb1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
C*b
1dAsdTsdTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1
Tb
1dTsdGsdCsdAsdAsdAsdAsdTsTb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1dTsdTsdGsdCsdAsdAsdAsdAsTb1Tb1C*b1Ab1Gb1
Ab
1
C*b
1
Ab
1
Tb
1
Tb
1dGsdCsdAsdAsdAsdAsdTsdTsC*b1Ab1Gb1
Ab
1
ssC*b
1
ssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
Ab
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
Ab
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssdTssdCss
Ab
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssAb
1
ssTb
1
ssdTssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Ab
1
ssC*b
1
ssdAssdTssdTssdGssdCssdAssdAssdAssdAssdTssdTss
C*b
1
ssAb
1
ssGb
1
Ab
1
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The antisense-oligonucleotides of the present invention are preferably administered in form of their pharmaceutically active salts optionally using substantially nontoxic pharmaceutically acceptable carriers, excipients, adjuvants, solvents or diluents. The medications of the present invention are prepared in a conventional solid or liquid carrier or diluents and a conventional pharmaceutically-made adjuvant at suitable dosage level in a known way. The preferred preparations and formulations are in administrable form which is suitable for infusion or injection (intrathecal, intracerebroventricular, intracranial, intravenous, intraparenchymal, intratumoral, intra- or extraocular, intraperitoneal, intramuscular, subcutaneous), local administration into the brain, inhalation, local administration into a solid tumor or oral application. However also other application forms are possible such as absorption through epithelial or mucocutaneous linings (oral mucosa, rectal and vaginal epithelial linings, nasopharyngial mucosa, intestinal mucosa), rectally, transdermally, topically, intradermally, intragastrically, intracutaneously, intravaginally, intravasally, intranasally, intrabuccally, percutaneously, sublingually application, or any other means available within the pharmaceutical arts.
The administrable formulations, for example, include injectable liquid formulations, retard formulations, powders especially for inhalation, pills, tablets, film tablets, coated tablets, dispersible granules, dragees, gels, syrups, slurries, suspensions, emulsions, capsules and deposits. Other administratable galenical formulations are also possible like a continuous injection through an implantable pump or a catheter into the brain.
As used herein the term “pharmaceutically acceptable” refers to any carrier which does not interfere with the effectiveness of the biological activity of the antisense-oligonucleotides as active ingredient in the formulation and that is not toxic to the host to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and the active compound can be administered to the subject at an effective dose.
An “effective dose” refers to an amount of the antisense-oligonucleotide as active ingredient that is sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology. An “effective dose” useful for treating and/or preventing these diseases or disorders may be determined using methods known to one skilled in the art. Furthermore, the antisense-oligonucleotides of the present invention may be mixed and administered together with liposomes, complex forming agents, receptor targeted molecules, solvents, preservatives and/or diluents.
Preferred are pharmaceutical preparations in form of infusion solutions or solid matrices for continuous release of the active ingredient, especially for continuous infusion for intrathecal administration, intracerebroventricular administration or intracranial administration of at least one antisense-oligonucleotide of the present invention. Also preferred are pharmaceutical preparations in form of solutions or solid matrices suitable for local administration into the brain. For fibrotic diseases of the lung, inhalation formulations are especially preferred.
A ready-to-use sterile solution comprises for example at least one antisense-oligonucleotide at a concentration ranging from 1 to 100 mg/ml, preferably from 5 to 25 mg/ml and an isotonic agent selected, for example, amongst sugars such as sucrose, lactose, mannitol or sorbitol. A suitable buffering agent, to control the solution pH to 6 to 8 (preferably 7-8), may be also included. Another optional ingredient of the formulation can be a non-ionic surfactant, such as Tween 20 or Tween 80.
A sterile lyophilized powder to be reconstituted for use comprises at least one antisense-oligonucleotide, and optionally a bulking agent (e.g. mannitol, trehalose, sorbitol, glycine) and/or a cryoprotectent (e.g. trehalose, mannitol). The solvent for reconstitution can be water for injectable compounds, with or without a buffering salt to control the pH to 6 to 8.
Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
A particularly preferred pharmaceutical composition is a lyophilized (freeze-dried) preparation (lyophilisate) suitable for administration by inhalation or for intravenous administration. To prepare the preferred lyophilized preparation at least one antisense-oligonucleotide of the invention is solubilized in a 4 to 5% (w/v) mannitol solution and the solution is then lyophilized. The mannitol solution can also be prepared in a suitable buffer solution as described above.
Further examples of suitable cryo-/lyoprotectants (otherwise referred to as bulking agents or stabilizers) include thiol-free albumin, immunoglobulins, polyalkyleneoxides (e.g. PEG, polypropylene glycols), trehalose, glucose, sucrose, sorbitol, dextran, maltose, raffinose, stachyose and other saccharides (cf. for instance WO 97/29782), while mannitol is used preferably. These can be used in conventional amounts in conventional lyophilization techniques. Methods of lyophilization are well known in the art of preparing pharmaceutical formulations.
For administration by inhalation the particle diameter of the lyophilized preparation is preferably between 2 to 5 μm, more preferably between 3 to 4 μm. The lyophilized preparation is particularly suitable for administration using an inhalator, for example the OPTINEB® or VENTA-NEB® inhalator (NEBU-TEC, Elsenfeld, Germany). The lyophilized product can be rehydrated in sterile distilled water or any other suitable liquid for inhalation administration. Alternatively, for intravenous administration the lyophilized product can be rehydrated in sterile distilled water or any other suitable liquid for intravenous administration.
After rehydration for administration in sterile distilled water or another suitable liquid the lyophilized preparation should have the approximate physiological osmolality of the target tissue for the rehydrated peptide preparation i.e. blood for intravenous administration or lung tissue for inhalation administration. Thus it is preferred that the rehydrated formulation is substantially isotonic.
The preferred dosage concentration for either intravenous, oral, or inhalation administration is between 10 to 2000 μmol/ml, and more preferably is between 200 to 800 μmol/ml.
For oral administration in the form of tablets or capsules, the at least one antisense-oligonucleotide may be combined with any oral nontoxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like. Moreover, when desired or needed, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture. Powders and tablets may be comprised of from about 5 to about 95 percent inventive composition.
Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethyl-cellulose, polyethylene glycol and waxes. Among the lubricants that may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include starch, methylcellulose, guar gum and the like.
Additionally, the compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of the at least one antisense-oligonucleotide to optimize the therapeutic effects. Suitable dosage forms for sustained release include implantable biodegradable matrices for sustained release containing the at least one antisense-oligonucleotide, layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the at least one antisense-oligonucleotide.
Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions.
Suitable diluents are substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol, starches derived from wheat, corn rice and potato, and celluloses such as microcrystalline cellulose. The amount of diluents in the composition can range from about 5% to about 95% by weight of the total composition, preferably from about 25% to about 75% by weight.
The term disintegrants refers to materials added to the composition to help it break apart (disintegrate) and release the medicaments. Suitable disintegrants include starches, “cold water soluble” modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures. The amount of disintegrant in the composition can range from about 1 to about 40% by weight of the composition, preferably 2 to about 30% by weight of the composition, more preferably from about 3 to 20% by weight of the composition, and most preferably from about 5 to about 10% by weight.
Binders characterize substances that bind or “glue” powders together and make them cohesive by forming granules, thus serving as the “adhesive” in the formulation. Binders add cohesive strength already available in the diluents or bulking agent. Suitable binders include sugars such as sucrose, starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcellulose and sodium carboxymethylcellulose and hydroxypropyl-methylcellulose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate. The amount of binder in the composition can range from about 1 to 30% by weight of the composition, preferably from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.
Lubricant refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear. Suitable lubricants include metallic stearates, such as magnesium stearate, calcium stearate or potassium stearate, stearic acid; high melting point waxes; and water soluble lubricants, such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and D,L-leucine. Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press. The amount of lubricant in the composition can range from about 0.05 to about 15% by weight of the composition, preferably 0.2 to about 5% by weight of the composition, more preferably from about 0.3 to about 3%, and most preferably from about 0.3 to about 1.5% by weight of the composition.
Glidants are materials that prevent caking and improve the flow characteristics of granulations, so that flow is smooth and uniform. Suitable glidants include silicon dioxide and talc. The amount of glidant in the composition can range from about 0.01 to 10% by weight of the composition, preferably 0.1% to about 7% by weight of the total composition, more preferably from about 0.2 to 5% by weight, and most preferably from about 0.5 to about 2% by weight.
In the pharmaceutical compositions disclosed herein the antisense-oligonucleotides are incorporated preferably in the form of their salts and optionally together with other components which increase stability of the antisense-oligonucleotides, increase recruitment of RNase H, increase target finding properties, enhance cellular uptake and the like. In order to achieve these goals, the antisense-oligonucleotides may be chemically modified instead of or in addition to the use of the further components useful for achieving these purposes. Thus the antisense-oligonucleotides of the invention may be chemically linked to moieties or components which enhance the activity, cellular distribution or cellular uptake etc. of the antisense-oligonucleotides. Such moieties include lipid moieties such as a cholesterol moiety, cholic acid, a thioether, hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid such as dihexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantine acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. The present invention also includes antisense-oligonucleotides which are chimeric compounds. “Chimeric” antisense-oligonucleotides in the context of this invention, are antisense-oligonucleotides, which contain two or more chemically distinct regions, one is the oligonucleotide sequence as disclosed herein which is connected to a moiety or component for increasing cellular uptake, increasing resistance to nuclease degradation, increasing binding affinity for the target nucleic acid, increasing recruitment of RNase H and so on. For instance, the additional region or moiety or component of the antisense-oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA hybrids or RNA:RNA molecules. By way of example, RNase H is a cellular endoribonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target which is the mRNA coding for the TGF-RII, thereby greatly enhancing the efficiency of antisense-oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used.
The present invention relates to the use of the antisense-oligonucleotides disclosed herein for suppression of ephrin-B2 function or for renal protective effects or for controlling nephrin function.
The present invention relates to the use of the antisense-oligonucleotides disclosed herein for use in the prophylaxis and treatment of nephropathy. The present invention relates to the use of the antisense-oligonucleotides disclosed herein for use in the prophylaxis and treatment of diabetic nephropathy. The present invention relates to the use of the antisense-oligonucleotides disclosed herein for use in the prophylaxis and treatment of proteinuria in diabetes. and/or nephropathy. For the treatment of proteinuria in diabetes and/or diabetic nephropathy db/db mice with uninephrectomy (UNx) surgery will be used as a model animal of type 2 diabetes with kidney complications. The mice will receive 20 mg/kg KGW ASO in a volume of 1 ml/kg KGW i.p. from the age of 14 weeks, twice a week for maximum 4 weeks. Albumin uria and structure of the kidneys will be analyzed.
The present invention further relates to a method of treating an animal (or human or patient) having a disease selected from nephropathy and/or diabetic proteinuria and/or diabetic nephropathy comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, and the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferred antisense-oligonucleotides comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2) are disclosed herein.
The present invention further relates to a method of treating an animal (or human or patient) having a disease selected from nephropathy and/or diabetic proteinuria and/or diabetic nephropathy comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, and the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide,
wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferred antisense-oligonucleotides comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1) are disclosed herein.
The present invention further relates to a method of inhibiting the expression of Ephrin-B2 in cells or tissues comprising incubating said cells or tissues with an effective amount of an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, and the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferred antisense-oligonucleotides comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2) are disclosed herein.
The present invention further relates to a method of inhibiting the expression of Ephrin-B2 in cells or tissues comprising incubating said cells or tissues with an effective amount of an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, and the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferred antisense-oligonucleotides comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1) are disclosed herein.
The present invention further relates to a method of restoring nephrin function in cells or tissues comprising incubating said cells or tissues with an effective amount of an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, and the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferred antisense-oligonucleotides comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence CTGAATTTTGCAATGT (Seq. ID No. 3) or AAATGCCTTGCTTGTA (Seq. ID No. 2) are disclosed herein.
The present invention further relates to a method of restoring nephrin function in cells or tissues comprising incubating said cells or tissues with an effective amount of an antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, and the antisense-oligonucleotide is capable of hybridizing with a region of the gene encoding Efnb2, or with a region of the mRNA encoding Efnb2, wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2, comprises the sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and the antisense-oligonucleotide comprises a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1), and salts and optical isomers of said antisense-oligonucleotide, wherein the region of the gene encoding Efnb2 is within an exon region of the gene encoding Efnb2; and wherein the region of the gene encoding Efnb2, or the region of the mRNA encoding Efnb2 comprises a sequence of at least 28 consecutive nucleotides that is 100% conserved between human and mouse. Preferred antisense-oligonucleotides comprising a sequence of at least 10 consecutive nucleotides capable of hybridizing with said sequence AATTCAGCCCTAACCT (Seq. ID No. 1) are disclosed herein.
Homo sapiens ephrin B2 (EFNB2), transcript variant 1
Homo sapiens ephrin B2 (EFNB2), transcript variant 2
Homo sapiens ephrin B2 (EFNB2), transcript variant 3
Homo sapiens
The Seq. ID No. 98 represents Homo sapiens ephrin B2 (EFNB2), transcript variant 1, mRNA (NCBI Reference Sequence: NM_004093.4) written in the DNA code, i.e. represented in G, C, A, T code, and not in the RNA code. The Seq. ID No. 99 represents Homo sapiens ephrin B2 (EFNB2), transcript variant 2, mRNA (NCBI Reference Sequence: NM_001372056.1) written in the DNA code, i.e. represented in G, C, A, T code, and not in the RNA code. The Seq. ID No. 100 represents Homo sapiens ephrin B2 (EFNB2), transcript variant 1, mRNA (NCBI Reference Sequence: NM_001372057.1) written in the DNA code, i.e. represented in G, C, A, T code, and not in the RNA code. The Seq. ID No. 101 represents the Homo sapiens chromosome 13, GRCh38.p13 Primary Assembly (NCBI Reference Sequence: NC_000013.11) (NC_000013.11:c106535662-106489745 Homo sapiens chromosome 13, GRCh38.p13 Primary Assembly). The Seq. ID No. 189 represents Mus musculus strain C57BL/6J chromosome 8, GRCm38.p6 C57BL/6J NCBI Reference Sequence: NC_000074.6.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
Oligonucleotides having the following sequences were used as references:
Male mice were used for all experiments. Cdh5-Cre ERT2 mice were bred with Efnb2 loxP-flanked homozygous mice (Efnb2lox/lox). The Efnb2lox/lox-Cdh5-Cre ERT2 negative mice were used as control littermates. To induce the genetic silencing of ephrin-B2, Tamoxifen (Sigma, T5648) was injected intraperitoneally at a dose of 50 μl of 3 mg/ml in Efnb2iΔEC and control littermates for three consecutive days from P21 to P23. To examine the specific expression of Cre in the glomerulus, Cdh5-Cre ERT2 mice were bred with R26-stop-EYFP mutant mice.
Experiments involving animals were conducted in accordance with institutional guidelines and laws, and following the protocols approved by the local animal ethics committees and authorities (Regierungspraesidium Darmstadt).
LOX is the name of cell line, isolated endothelial cells from ephrin-B2 floxed/floxed mice carrying T antigen. Then the cells were infected the plasmid DNA encoding Cre to KO Efnb2, the gene encoding ephrin-B2.
To induce diabetes, Streptozotocin (Sigma, SO130) dissolved in Sodium-Citrate buffer, pH 4.5 (Sigma, C8532) and at 40 mg/Kg body weight was intraperitoneally injected from P35 to P39. The mice were provided with 10% sucrose water (Sigma, SO389) to prevent and treat hypoglycemia when required. Subsequent to Streptozotocin injections, the mice were fed with western-type high fat diet until the end point of the experiments (ssniff Spezialdiäten GmbH, E15126-34) to aid in the development of diabetes. Mice were checked for blood glucose values prior to and at the end of the protocol by drawing blood via the tail-vein. The control non-diabetic mice were only fed the western-type high fat diet from 5 weeks of age until 18 weeks of age.
Mouse kidney sections were prepared by cryosectioning (10 μm thickness) and fixed for 10 min using 4% paraformaldehyde solution in PBS (pH 7.4). Fixed sections were washed 3× with PBS and incubated overnight at 4° C. with the appropriate primary antibodies against ephrin-B2 (Sigma, HPA0008999, dilution 1:100), CD31 (BD Pharmingen, 557355, dilution 1:100), Nephrin (R&D Systems, AF3159, dilution 1:100), phospho-EphB4 (Signalway Antibody, 12720, dilution 1:100), phospho-Nephrin (Abcam, ab80299, dilution 1:100), or GFP (Invitrogen, A21311, dilution 1:200). After incubation with the primary antibodies, the sections were washed 3× with PBS and incubated for 2 hours at room temperature with Biotin-SP-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories Inc., 705-065-147, dilution 1:200) and additional relevant Alexa Fluor secondary antibodies. The sections were washed 3× with PBS and incubated for an additional two hours with streptavidin Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific, Invitrogen, Life Technologies) for biotin. Following washing 3× with PBS, sections were counter-stained with DAPI (Thermo Fisher Scientific, Invitrogen, Life Technologies; D3571) solution in PBS for 15 min. Sections were washed and mounted using Fluoromount-G® mounting medium (SouthernBiotech, 0100-01).
Specimens for human study analysis were taken from patients after informed consent. The use of these specimens was approved by the IRB of the Washington University School of Medicine, in adherence to the Declaration of Helsinki.
Formaldehyde-fixed paraffin-embedded kidney tissue samples were deparafinized, and antigen retrieval was performed by boiling the slides in EDTA buffer (pH 8) for 15 min, blocked with 5% BSA/5% FBS/0.1% Tween-20 for 30 min, and treated with rabbit anti-ephrin-B2 antibody (Sigma, HPA0008999, dilution 1:100) and goat anti-Nephrin antibody (R&D Systems, AF3159, dilution 1:100) at 4° C. overnight. Then slides were incubated with AlexaFluor 546 donkey anti-goat IgG (1:500 dilution; Invitrogen), AlexaFluor 488 donkey anti-rabbit IgG, and Hoechst 33342 (1:500 dilution; Thermo Fisher Scientific) at room temperature for 2 hours. Images were taken using a Leica SP8 microscope. To measure mean signal intensity of the pixels in the glomerulus, Velocity (Improvision) was used for quantitative image analysis. Photoshop CS, and Illustrator CS (Adobe) software were used for image processing and in compliance with general guidelines for image processing.
Kidneys collected at experimental end-point were dehydrated and paraffin mounted. Following sectioning using a microtome (3-4 μm thick), slides were placed at 60° C. to enable the paraffin to melt and fix the tissue onto the slide. The kidney sections were then re-hydrated by immersion in xylene (10 min, 3×), 100% ethanol (5 min, 3×), 95% ethanol (5 min, 1×), 80% ethanol (5 min, 1×) and de-ionized water (5 minutes, 1×) sequentially. The sections were immersed in hemotoxylin (Waldeck; 2E-038) solution for 5 min, after which they were washed with running tap water till the appropriate levels of blue color corresponding to hemotoxylin had developed. Following this, sections were placed in eosin (Waldeck; 2C-140) solution for 1 min and washed 3× in 95% ethanol for 3 min. Stained kidney sections were again dehydrated in 100% ethanol (3 min, 3×) and xylene (5 min, 3×) followed by mounting using the Entellan® New (Merck Millipore; 107961) mounting medium.
For EM, small pieces (transmission EM) of kidney tissue were fixed in 4% paraformaldehyde and 0.2% gluteraldehyde overnight Samples were incubated with 20% donkey serum and 0.1% photo-flo (Electron Microscopy Sciences) in PBS for 30 min and then were incubated with an anti-Nephrin antibody (R&D systems, AF3159 dilution 1:100) at 4° C. overnight followed by rabbit anti-goat Fab′ (Nanoprobes, 2006, dilution 1:100) at 4° C. overnight. After washing in 0.1M phosphate buffer and fixing with 1% glutaraldehyde, signal was enhanced with HQ Silver enhancement kit (Nanoprobes 2012). After refixing with OsO4, the samples were dehydrated in a graded ethanol series and embedded in epoxy resin. Ultrathin sections were examined by EM (Hitachi, H-7650)
Urine albumin values and urine creatinine values were determined using the Mouse Albumin ELISA Kit (Shibayagi Co.Ltd, AKRAL-121) and Creatinine Assay Kit (AbCam, ab65340) according to the manufacturers instruction. The optical density (O.D.) read-out of the ELISA plates was measured at 450 nm for albumin values and at 570 nm for creatinine values using a 96-well plate reader. The UACR values were obtained by dividing the final values of albumin in milligrams/liter (mg/l) by the creatinine values obtained in grams/liter (g/l) for each time point per mouse. The serum creatinine values were determined using a Creatinine Assay Kit (AbCam, ab65340) as described above. Serum stored at -80° C. was thawed on ice and used for the assay without any dilution.
Western Blotting Analysis with Kidney Lysate
Collected whole kidney tissue was weighed, cut into small pieces and suspended in 3 mL/gram tissue of RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.5% Na-deoxycholate; 0.1% SDS; 150 mM NaCl; 2 mM EDTA; Protease inhibitor (Sigma, P2714, 1:100)) and homogenized with a hand-held sonicator. The samples were then centrifuged at 10000×g for 10 min. The supernatant was transferred into a new tube and spun again at 10000×g for 30 min at 4° C. The supernatant was then used for further analysis. Protein concentration was determined using a BCA protein assay (Pierce) and 2 mg of protein was suspended in 3×SDS loading buffer, boiled and used for western blot analysis using anti-Nephrin (R&D systems, AF3159, dilution 1:1000) and anti-a-tubulin (Sigma, T5168, 1:20000) antibodies. Signal intensity was measured with ImageJ.
Immunoprecipitation of EphB4 from Kidney Lysates
Kidney lysates were prepared as for western blotting, then incubated with 2 μg of an anti-EphB4 antibody (R&D systems, MAB446) for 1 hr at 4° C. with rotation. Then 20 μl of Dynabeads G (Thermo Fisher Scientific, Invitrogen, Life Technologies; 10004D), pre-washed with wash buffer (50 mM Tris-HCl pH 8.0, EDTA 1 mM, NaCl 150 mM, NP-40 1%, Protease inhibitor (Sigma, P2714, 1:100) and Phosphatase inhibitor (Merck Millipore, 524625)), were added. After 1 hr incubation at 4° C., the Dynabeads G were separated using a magnetic separator and washed 5× with wash buffer. The beads were re-suspended in 30 μl of 1×SDS sample buffer for western blotting analysis using anti-Nephrin (R&D Systems, AF3159, dilution 1:1000) and anti-EphB4 (R&D systems, AF446, 1:1000) antibodies.
Immunoprecipitation of Ephrin-B2 from Cultured ECs
Efnb2 lox/lox cells and KO cells (1×106 cells/10-cm dish)15 were seeded and incubated for 24 hours. Cells were washed with ice-cold PBS and lysed with 800 μl of lysis buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 150 mM NaCl, protease inhibitor cocktail (Sigma, P2714, 1:100), phosphatase inhibitor cocktail (Calbiochem, 524629, 1:50). The lysates were centrifuged at 20000×g for 10 min at 4° C., and the supernatant was incubated with protein G sepharose beads crosslinked with 2 μg of ephrin-B2 antibody (R&D systems AF 496) at 4° C. for 1 hr. The beads were washed three times with an excess of lysis buffer and eluted with 60 μl of 1×SDS sample buffer. Then, 20 μl of each eluate was subjected to SDS-PAGE, followed by immunoblotting with anti-ephrin-B2 antibody (Sigma, HPA008999).
Transfection of cDNA
pEGFP (clontech) pBabe-puro-GFP-flag-ephrin-B2, pcDNA3-HA-Capnsl (kindly gifted by Dr. Claudio Schneider), or pCMV-Rbpj-myc-Flag (kindly provided by the Lead Discovery Center GmbH, Dortmund) were transfected into HEK293 cells with Polyethylenimine (PEI; Polysciences Inc., 24765). 200 μl of transfection mix was prepared by mixing 100 μl of 150 mM NaCl solution in distilled water with 2 μg plasmid DNA, followed by vortexing the mix and subsequently addition of 100 μl of PEI-NaCl solution. The PEI-NaCl-plasmid transfection mix was incubated for 10 min and was added to cells. The transfection mix was incubated with cells for 6-8 hours. The cells were washed and fresh cell culture medium was added. For HUVECs, jet-PEI® DNA Transfection reagent (PolyPlus, 101-10) was used according to the manufacturers instructions.
48 hours after transfection, cells were lysed with lysis buffer (20 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1 mM DTT, 1% CHAPS, 150 mM NaCl and 1× Protease inhibitor (Sigma, P2714)). Cell lysate or the medium was clarified by centrifugation at 10000×g for 10 min at 4° C. The supernatants were incubated with Flag-M2 magnetic beads (Sigma, M8823) for 2 hours at 4° C. The Flag-M2 magnetic beads were separated using a magnetic separator and washed 3 times with lysis buffer. The beads were suspended in SDS sample buffer for western blotting analysis using anti-Flag-M2 HRP conjugated (Sigma, A8592, dilution 1:10,000), anti-ephrin-B2 (Sigma, HPA008999, dilution 1:1000) or anti-HA-HRP conjugated (Cell Signaling Technology, 2999 dilution 1:10000) antibodies.
ASOs were transfected into Lox cells with TransIT-X2® Dynamic Delivery System (Mirus Bio, Madison, WI). 50 μL of ASO mix was prepared by mixing 6 μl of ASO in Opti-MEM, followed by vortexing the mix and subsequently addition of 3 μl of TransIT-X2. The ASO transfection mix was incubated for 30 min and was added to cells (final concentration 100 nM). The transfection mix was incubated with cells for 48 hours.
mRNA was extracted with Quick-RNA Miniprep Kit (R1054, Zymo Research, Freiburg Germany). Reverse transcription was performed with SuperScript IV VILO (11756050, ThermoFisherScientific, MA). quantitative PCR was performed with Power SYBR Green Master Mix (4367659, Thermo Fisher Scientific) with following primers: mouse beta-actin_Fwd 5′-GAAATCGTGCGTGACATCAAAG-3′, mouse beta-actin_Rvr 5′-TGTAGTTTCATGGATGCCACAG-3′, mouse Efnb2-1_Fwd 5′-ATTATTTGCCCCAAAGTGGACTC-3′, mouse Efnb2-1_Rvs 5′-GCAGCGGGGTATTCTCCTTC-3′
Result for antisense oligonucleotide Seq. ID 74p are shown in
Free uptake 10, 3.3, 1.1, 0.37, 0.12 μM
The antisense-oligonucleotides in form of gapmers were prepared on an ABI 394 synthesizer (Applied Biosystems) according to the phosphoramidite oligomerization chemistry using 500 A controlled pore glass (CPG) loaded with Unylinker™ Chemgenes (Wilmington, MA, USA) as solid support to give a 3 μmol synthesis scale.
Alternatively, the antisense-oligonucleotides can be prepared on an ABI 3900 or an Expedite™ (Applied Biosystems) according to the phosphoramidite oligomerization chemistry. On the AB13900, the solid support can be polystyrene loaded with UnySupport (Glen Research, Sterling, Virginia, USA) to give a synthesis scale of 0.2 μmol.
As DNA building-units 5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-N4-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite and 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite were used, which correspond to the 5′-O-(4,4′-dimethoxytrityl)-2′-O,3′-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite monomers of deoxy thymidine (dT), 4-N-benzoyl-2′-deoxy-cytidine (dCBz), 6-N-benzoyl-2′-deoxy-adenosine (dABz) and 2-N-isobutyryl-2′-deoxy-guanosine (dGiBu)
As LNA-building-units (β-D-oxy-LNA) 5′-O-DMT-2′-O,4′-C-methylene-N2-dimethylformamidine-guanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (LNA-GDMF), 5′-O-DMT-2′-O,4′-C-methylene-thymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (LNA-T), 5′-O-DMT-2′-O,4′-C-methylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (LNA-ABz), 5′-O-DMT-2′—O—,4′-C-methylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (LNA-C*Bz) were used.
The phosphoramidites of the LNAs were dissolved in dry acetonitrile to give 0.07 M-oligonucleotide except LNA-C*Bz which was dissolved in a mixture of THE/acetonitrile (25/75 (v/v)). In case of LNA-ABz (MW=885.9 g/mole, CAS No. [206055-79-0]) 100 mg were dissolved in 1.6 ml anhydrous acetonitrile. In case of LNA-C*Bz (MW=875.9 g/mol, CAS No. [206055-82-5]) 100 mg were dissolved in 1.6 ml THE/acetonitrile 25/75 (v/v). In case of LNA-GDMF (MW=852.9 g/mol, CAS No. [709641-79-2]) 100 mg were dissolved in 1.7 ml anhydrous acetonitrile. In case of LNA-T (MW=772.8 g/mol, CAS No. [206055-75-6]) 100 mg was dissolved in 1.8 ml anhydrous acetonitrile.
The β-D-thio-LNAs 5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-N6-benzoyladenosine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, 5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5′-O-DMT-2′-deoxy-2′-mercapto-2′-S,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were synthesized as described in J. Org. Chem. 1998, 63, 6078-6079.
The synthesis of the β-D-amino-LNA 5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-5-methyl-N4-benzoylcytidine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-N6-benzoyladenosine-3′-[(2-cyanoethyl-N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, 5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-O-DMT-2′-deoxy-2′-methylamino-2′-N,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5′-O-DMT-2′-deoxy-2′-amino-2′-N,4′-C-methylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites was carried out according to the literature procedure (J. Org Chem. 1998, 63, 6078-6079).
The α-L-oxy-LNAs α-L-5′-O-DMT-2′-O,4′-C-methylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, α-L-5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, α-L-5′-O-DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and α-L-5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were performed similarly to the procedures described in the literature (J. Am. Chem. Soc. 2002, 124, 2164-2176; Angew. Chem. Int. Ed. 2000, 39, 1656-1659).
The synthesis of β-D-ENA LNAs 5′-O-DMT-2′-O,4′-C-ethylene-N2-dimethylformamidine-guanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphor-amidite, 5′-O-DMT-2′-O,4′-C-ethylene-thymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite, 5′-O-DMT-2′-O,4′-C-ethylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, and 5′-O-DMT-2′-O—,4′-C-ethylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite was carried out according to the literature procedure (Nucleic Acids Research 2001, Supplement No. 1, 241-242).
The synthesis of β-D-ENA LNAs 5′-O-DMT-2′-O,4′-C-ethylene-N2-dimethylformamidine-guanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphor-amidite, 5′-O-DMT-2′-O,4′-C-ethylene-thymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite, 5′-O-DMT-2′-O,4′-C-ethylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, and 5′-O-DMT-2′-O—,4′-C-ethylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite was carried out according to the literature procedure (Nucleic Acids Research 2001, Supplement No. 1, 241-242).
The (β-(benzoylmercapto)ethyl)pyrrolidinolthiophosphoramidites for the synthesis of the oligonucleotide with phosphorodithioate backbone were prepared in analogy to the protocol reported by Caruthers (J. Org. Chem. 1996, 61, 4272-4281).
The “phosphoramidites-C3” (3-(4,4′-dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and the “3′-Spacer C3 CPG” (1-dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG were purchased.
5′-O-DMT-3′-hydroxy-nucleoside monomer, succinic anhydride (1.2 eq.) and DMAP (1.2 eq.) were dissolved in 35 ml dichloromethane (DCM). The reaction was stirred at room temperature overnight. After extractions with NaH2PO4 0.1 M pH 5.5 (2×) and brine (1×), the organic layer was further dried with anhydrous NaSO4 filtered and evaporated. The hemiester derivative was obtained in 95% yield and was used without any further purification.
The above prepared hemiester derivative (90 μmol) was dissolved in a minimum amount of DMF, DIEA and pyBOP (90 μmol) were added and mixed together for 1 min. This pre-activated mixture was combined with LCAA-CPG (500 A, 80-120 mesh size, 300 mg) in a manual synthesizer and stirred. After 1.5 hours at room temperature, the support was filtered off and washed with DMF, DCM and MeOH. After drying, the loading was determined to be 57 μmol/g.
4,5-Dicyanoimidazole (DCI) as described in WO2007/112754 was employed for the coupling of the phosphoramidites. Instead of DCI other reagents, such as 5-ethylthio-1H-tetrazole (ETT) (0.5 M in acetonitrile), 5-benzylthio-1H-tetrazole or saccharin-1-methylimidazol can be used as activator. 0.25 M DCI in acetonitrile was used for the coupling with LNA.
10% acetic anhydride (Ac2O) in THE (HPLC grade) and 10% N-methylimidazol (NMI) in THF/pyridine (8:1) (HPLC grade) were added and allowed to react.
Phosphorous(III) to Phosphorous(V) is normally done with e.g. iodine/THF/pyridine/H2O using 0.02 M iodine in THF/Pyridine/H2O or 0.5 M 1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO) in anhydrous acetonitrile.
In the case that a phosphorthioate internucleoside linkage is prepared, a thiolation step is performed using 0.2 M 3H-1,2-benzothiole-3-one 1,1-dioxide (Beaucage reagent) in anhydrous acetonitrile. The thiolation can also be carried out by using a 0.05 M solution of 3-((dimethylamino-methylidene)-amino)-3H-1,2,4-dithiazole-3-thione (DDTT) in anhydrous acetonitrile/pyridine (1:1 v/v) or by using xanthane chloride (0.01 M in acetonitrile/pyridine 10%) as described in WO2007/112754. Alternatively, other reagents for the thiolation step such as xanthane hydride (5-imino-(1,2,4)dithiazolidine-3-thione), phenylacetyl disulfide (PADS) can be applied.
In the case that a phosphordithioate internucleoside linkage is prepared, the resulting thiophosphite triester was oxidized to the phosphorothiotriester by addition of 0.05 M DDTT (3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione) in pyridine/acetonitrile (4:1 v/v).
Cleavage from the Solid Support and Deprotection
At the end of the solid phase synthesis, the final 5′-O-(4,4′-dimethoxytrityl) group can be removed on the synthesizer using the “Deblock” reagent or the group may be still present while the oligonucleotide is cleaved from the solid support. The DMT groups were removed with trichloroacetic acid.
Upon completion of the solid phase synthesis antisense-oligonucleotides were treated with a 20% diethylamine solution in acetonitrile for 20 min. to remove the cyanoethyl protecting groups on the phosphate backbone. Subsequently, the antisense-oligonucleotides were cleaved from the solid support and deprotected using 1 to 5 mL concentrated aqueous ammonia for 16 hours at 55° C. The solid support was separated from the antisense-oligonucleotides by filtration or centrifugation.
If the oligonucleotides contain phosphorodithioate triester, the thiol-groups were deprotected with thiophenol:triethylamine:dioxane, 1:1:2, v/v/v for 24 h, then the oligonucleotides were cleaved from the solid support using aqueous ammonia for 1-2 hours at room temperature, and further deprotected for 4 hours at 65° C.
Removal of 5′-O-(4,4′-Dimethoxytrityl) Group after Cleavage from Solid Support:
The oligonucleotides were cleaved from the solid support using aqueous ammonia for 1-2 hours at room temperature, and further deprotected for 4 hours at 65° C. The oligonucleotides were purified by reverse phase HPLC (RP-HPLC), and then the DMT-group is removed with trichloroacetic acid.
If the oligonucleotides contain phosphorodithioate triester, the cleavage from the solid-support and the deprotection of the thiol-group were performed by the addition of 850 μl ammonia in concentrated ethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h.
Terminal groups at the 5′-end of the antisense oligonucleotide:
The solid supported oligonucleotide was treated with 3% trichloroacetic acid in dichloromethane (w/v) to completely remove the 5′-DMT protection group. Further, the compound was converted with an appropriate terminal group with cyanoethyl-N,N-diisopropyl)phosphoramidite-moiety. After the oxidation of the phosphorus(III) to phosphorus(V), the deprotection, detachment from the solid support and deprotection sequence were performed as described above.
The crude antisense-oligonucleotides were purified by anion-exchange high-performance liquid chromatography (HPLC) on an AKTA Explorer chromatography system (GE Healthcare, Freiburg, Germany) and a column packed with Source Q15 (GE Healthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and buffer B was the same as buffer A with the exception of 500 mM sodium perchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV) was employed. UV traces at 280 nm were recorded. Appropriate fractions were pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol. Finally, the pellet was washed with 70% ethanol.
Identity of the antisense-oligonucleotides was confirmed by electrospray ionization mass spectrometry (ESI-MS) and purity was determined by analytical OligoPro Capillary Electrophoresis (CE).
The purification of the dithioate was performed on an Amersham Biosciences P920 FPLC instrument fitted with a Mono Q 10/100 GL column. The buffers were prepared with DEPC-treated water, and their compositions were as follows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.
5′-O-DMT-2′-O,4′-C-methylene-thymidine-3′-O-succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group. After several washes with a total amount of 800 μl acetonitrile, the coupling reaction was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N-methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N4-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N4-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-O—,4′-C-methylene-5-methyl-N4-benzoylcytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
Upon completion of the solid phase synthesis, the antisense-oligonucleotides were treated with a 20% diethylamine solution in acetonitrile for 20 min. to remove the cyanoethyl protecting groups on the phosphate backbone.
Subsequently, the antisense-oligonucleotides were cleaved from the solid support and further deprotected using 5 mL concentrated aqueous ammonia for 16 hours at 55° C. The solid support was separated from the antisense-oligonucleotides by filtration or centrifugation.
The crude antisense-oligonucleotide was purified by anion-exchange high-performance liquid chromatography (HPLC) according to the general procedure as described above.
5′-O-DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine 3′-O succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group. After several washes with a total amount of 800 μl acetonitrile, the coupling reaction was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-N6-benzoyladenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N4-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N4-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-thymidine 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazole (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
Upon completion of the solid phase synthesis, the antisense-oligonucleotides were treated with a 20% diethylamine solution in acetonitrile (Biosolve BV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethyl protecting groups on the phosphate backbone.
Subsequently, the antisense-oligonucleotides were cleaved from the solid support and further deprotected using 5 mL concentrated aqueous ammonia for 16 hours at 55° C. The solid support was separated from the antisense-oligonucleotides by filtration or centrifugation.
The crude antisense-oligonucleotides were purified by anion-exchange high-performance liquid chromatography (HPLC) on an AKTA Explorer System (GE Healthcare, Freiburg, Germany) and a column packed with Source Q15 (GE Healthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and buffer B was the same as buffer A with the exception of 500 mM sodium perchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV) was employed. UV traces at 280 nm were recorded. Appropriate fractions were pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol. Finally, the pellet was washed with 70% ethanol.
5′-O-DMT-2′-O,4′-C-methylene-N6-benzoxyladenosine-3′-O-succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group. After several washes with a total amount of 800 μl acetonitrile, the coupling reaction was carried out with 80 μl 5′ O DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N4-benzoyl-2′-deoxycytidine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N2-isobutyryl-2′-deoxyguanosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-N6-benzoyl-2′-deoxyadenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-deoxythymidine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloro-methane for 60 s to completely remove the 5′-DMT protection group.
The coupling was carried out with 80 μl 5′-O-DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (0.07 M) and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 640 μl of Beaucage (0.2 M) were inserted to the column for 180 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THF (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
Upon completion of the solid phase synthesis, the antisense-oligonucleotides were treated with a 20% diethylamine solution in acetonitrile (Biosolve BV, Valkenswaard, The Netherlands) for 20 min. to remove the cyanoethyl protecting groups on the phosphate backbone.
Subsequently, the antisense-oligonucleotides were cleaved from the solid support and further deprotected using 5 mL concentrated aqueous ammonia for 16 hours at 55° C. The solid support was separated from the antisense-oligonucleotides by filtration or centrifugation.
The crude antisense-oligonucleotides were purified by anion-exchange high-performance liquid chromatography (HPLC) on an AKTA Explorer System (GE Healthcare, Freiburg, Germany) and a column packed with Source Q15 (GE Healthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 and contained 20% acetonitrile and buffer B was the same as buffer A with the exception of 500 mM sodium perchlorate. A gradient of 15% B to 55% B within 32 column volumes (CV) was employed. UV traces at 280 nm were recorded. Appropriate fractions were pooled and precipitated with 3 M NaOAc, pH=5.2 and 70% ethanol. Finally, the pellet was washed with 70% ethanol.
The antisense oligonucleotides of Seq. ID No. 15m, 17m, 18m, 21i, 22i, 23i, 21j, 22j, 23j, 26m, 27m, 28m, 29m, 26n, 27n, 28n, 29n, 26o, 27o, 28o, 29o, 30q, 31q, 32q, 30r, 31r, 32r, 30s, 31s, 32s, 30t, 31t, 32t, 19q, 20q, 19r, 20r, 19s, 20s, 19t, 20t, 4as, 4at, 4au, 4av, 4aw, 4ax, 4ay, 4az, 4ba, 4bb and 4bc were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.3.
The antisense oligonucleotides of Seq. ID No. 45m, 47m, 48m, 44m, 52i, 53i, 54i, 55i, 52j, 53j, 54j, 55j, 57m, 58m, 60m, 59m, 57n, 58n, 60n, 59n, 57o, 58o, 60o, 59o, 61q, 62q, 63q, 61r, 62r, 63r, 61s, 62s, 63s, 61t, 62t, 63t, 49q, 50q, 49r, 50r, 49s, 50s, 49t, 50t, 5as, 5at, 5au, 5av, 5aw, 5ax, 5ay, 5az, 5ba, 5bb and 5bc were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.1.
The antisense oligonucleotides of Seq. ID No. 74m, 77m, 78m, 84i, 85i, 86i, 84j, 85j, 86j, 88m, 89m, 90m, 88n, 89n, 90n, 88o, 89o, 90o, 91q, 92q, 93q, 91r, 92r, 93r, 91s, 92s, 93s, 91t, 92t, 93t, 79q, 80q, 79r, 80r, 79s, 80s, 79t, 80t, 6as, 6at, 6au, 6av, 6aw, 6ax, 6ay, 6az, 6ba, 6bb, 6bc were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.2.
The LNA was bound to CPG according to the general procedure. The coupling reaction and capping step were also carried out as described in example 3.3.
The antisense oligonucleotides of Seq. ID No. 15p, 17p, 18p, 21k, 22k, 23k, 21l, 22l, 23l, 9f, 10f, 11f, 26p, 27p, 28p, 29p, 26q, 27q, 28q, 29q, 26r, 27r, 28r, 29r, 30u, 31u, 32u, 30v, 31v, 32v, 30w, 31w, 32w, 30x, 31x, 32x, 19u, 20u, 19v, 20v, 19w, 20w, 19x, 20x, 4bd, 4be, 4bf, 4bg, 4bh, 4bi, 4bj, 4bk, 4bl, 4bm and 4bn were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.3.
The antisense oligonucleotides of Seq. ID No. 46p, 45p, 47p, 48p, 44p, 52k, 53k, 54k, 55k, 52l, 53l, 54l, 55l, 39f, 40f, 41f, 57p, 58p, 60p, 59p, 57q, 58q, 60q, 59q, 57r, 58r, 60r, 59r, 61u, 62u, 63u, 61v, 62v, 63v, 61w, 62w, 63w, 61x, 62x, 63x, 49u, 50u, 49v, 50v, 49w, 50w, 49x, 50x, 5bd, 5be, 5bf, 5bg, 5bh, 5bi, 5bj, 5bk, 5bl, 5bm and 5bn were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.1.
The antisense oligonucleotides of Seq. ID No. 74p, 77p, 78p, 84k, 85k, 86k, 84l, 85l, 86l, 71f, 72f, 73f, 88p, 89p, 90p, 88q, 89q, 90q, 88r, 89r, 90r, 91u, 92u, 93u, 91v, 92v, 93v, 91w, 92w, 93w, 91x, 92x, 93x, 79u, 80u, 79v, 80v, 79w, 80w, 79x, 80x, 6bd, 6be, 6bf, 6bg, 6bh, 6bi, 6bj, 6bk, 6bl, 6bm and 6bn were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.2.
Gb1ssGb1ssdTssdTssdAssdGssdGssdGssdCssdTssGb1ssAb1 (Seq. ID. 16s) 5′-O-DMT-2′-O,4′-C-methylene-N6-benzoxyladenosine-3′-O-succinoyl-linked LCAA CPG (0.2 μmol) was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group. After several washes with a total amount of 800 μl acetonitrile, the coupling reaction was carried out with 38 μl 5′-O-DMT-2′-O,4′-C-methylene-N2-dimethyformamidineguanosine-3′-[(3-benzoylmercapto)ethyl]pyrrolidinolthiophos-phoramidite (0.15 M) in 10% dichloromethane (v/v) in acetonitrile and 236 μl DCI in acetonitrile (0.25 M). The coupling reaction was allowed to take place for 250 sec., and excess reagents were flashed out with 800 μl acetonitrile, and 900 μl of DDTT (0.05 M in pyridine/acetonitrile 4:1 v/v) were inserted to the column for 240 s The system was flushed with 320 μl acetonitrile. For the capping step, 448 μl of acetic anhydride in THE (1:9 v/v) and 448 μl N methylimidazol (NMI)/THF/pyridine (1:8:1) were added and allowed to react for 45 sec. At the end of this cycle, the system was washed with 480 μl acetonitrile. The compound was treated with 1400 μl 3% trichloroacetic acid in dichloromethane for 60 s to completely remove the 5′-DMT protection group.
The further elongation of the oligonucleotide was performed in the same way as described in the previous examples.
Upon completion of the solid phase synthesis, the antisense-oligonucleotides were treated 850 μl ammonia in concentrated ethanol (ammonia/ethanol 3:1 v/v) at 55° C. for 15-16h in order to cleave antisense-oligonucleotide from the solid-support and to deprotect the thiol-group.
Next, the crude antisense-oligonucleotide was purified by anion-exchange chromatography using a Mono Q 10/100 GL column. The buffers were prepared with DEPC-treated water, and their compositions were as follows: Buffer A: 25 mM Tris-HCl, 1 mM EDTA, pH 8.0; Buffer B: 25 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH 8.0.
The antisense oligonucleotides of Seq. ID No. 15s, 17s, 18s, 21m, 22m, 23m, 21n, 22n, 23n, 9g, 10g, 11g, 26s, 27s, 28s, 29s, 26t, 27t, 28t, 29t, 26u, 27u, 28u, 29u, 30y, 31y, 32y, 30z, 31z, 32z, 30aa, 31aa, 32aa, 30ab, 31ab, 32ab, 19y, 20y, 19z, 20z, 19aa, 20aa, 19ab, 20ab, 4bo, 4 bp, 4bq, 4br, 4bs, 4bt, 4bu, 4bv, 4bw, 4bx, 4by, 46s, 45s, 47s, 48s, 44s, 52m, 53m, 54m, 55m, 52n, 53n, 54n, 55n, 39g, 40g, 41g, 57s, 58s, 60s, 59s, 57t, 58t, 60t, 59t, 57u, 58u, 60u, 59u, 61y, 62y, 63y, 61z, 62z, 63z, 61 aa, 62aa, 63aa, 61 ab, 62ab, 63ab, 49y, 50y, 49z, 50z, 49aa, 50aa, 49ab, 50ab, 5bo, 5 bp, 5bq, 5br, 5bs, 5bt, 5bu, 5bv, 5bw, 5bx, 5by, 74s, 77s, 78s, 84m, 85m, 86m, 84n, 85n, 86n, 71g, 72g, 73g, 88s, 89s, 90s, 88t, 89t, 90t, 88u, 89u, 90u, 91y, 92y, 93y, 91z, 92z, 93z, 91aa, 92aa, 93aa, 91ab, 92ab, 93ab, 79y, 80y, 79z, 80z, 79aa, 80aa, 79ab, 80ab, 6bo, 6 bp, 6bq, 6br, 6bs, 6bt, 6bu, 6bv, 6bw, 6bx and 6by were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.11.
The antisense oligonucleotides of Seq. ID No. 9e, 10e, 11e, 39e, 40e, 41e, 71e, 72e and 73e were synthesized according to the general procedure with the appropriate DNA and LNA building units as exemplified in example 3.11.
The other oligonucleotides of Seq. ID No. 16n, 15n, 17n, 18n, 46n, 45n, 47n, 48n, 44n, 74n, 77n, 78n, 16o, 15o, 17o, 18o, 46o, 45o, 47o, 48o, 44o, 74o, 77o, 78o, 16t, 15t, 17t, 18t, 46t, 45t, 47t, 48t, 44t, 74t, 77t, 78t, 16u, 15u, 17u, 18u, 46u, 45u, 47u, 48u, 44u, 74u, 77u, 78u, 16q, 15q, 17q, 18q, 46q, 45q, 47q, 48q, 44q, 74q, 77q, 78q, 16r, 15r, 17r, 18r, 46r, 45r, 47r, 48r, 44r, 74r, 77r and 78r were synthesized according to the general procedure and as shown in the previous examples. The preparation of the antisense-oligonucleotide including β-D-thio-LNA, β-D-amino-LNA, α-L-oxy-LNA, β-D-ENA, β-D-(NH)-LNA, or β-D-(NCH3)-LNA units were performed in the same way as the antisense-oligonucleotides containing β-D-oxy-LNA units.
| Number | Date | Country | Kind |
|---|---|---|---|
| EP 21 179 667.7 | Jun 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/066418 | 6/15/2022 | WO |
