nucleic acid molecule, method for synthesizing a tricyclic phosphorothioate DNA molecule and pharmac

专利摘要:
TRICYCLE-PHOSPHOROTIOATE DNA. The present invention relates to a nucleic acid molecule that contains a sequence of tricyclic nucleosides joined by means of internucleoside phosphorothioate linkage. The invention also relates to synthetic antisense oligonucleotides and methods that employ them.
公开号:BR112014009066B1
申请号:R112014009066-1
申请日:2012-10-12
公开日:2020-08-25
发明作者:Christian Leumann；Luis Garcia；Thomas Voit
申请人:Association Institut De Myologies；Universität Bern；Universite Pierre Et Marie Curie (Paris 6)；Institut National de la Santé et de la Recherche Médicale；Centre National De La Recherche Scientifique；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to a nucleic acid molecule that contains a sequence of tricyclonucleosides joined by internucleoside bonds. The invention also relates to synthetic antisense oligonucleotides and methods for employing them. BACKGROUND OF THE INVENTION
[0002] [0002] Tricycle-DNAs (tc-DNA) are a class of restricted DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the main chain and to optimize the main chain geometry of the angle torsion γ. tc-DNAs containing adenine and homobasic thymine form extraordinarily stable A-T base pairs with complementary RNAs.
[0003] [0003] Recently, the present inventors have proposed using the advantageous properties of this class of nucleic acids in antisense oligonucleotides for the treatment of various diseases. International patent application No. PCT / EP2010 / 054735 discloses synthetic antisense oligonucleotides and methods that employ antisense oligonucleotides to modify splicing events that occur during pre-mRNA processing or downwardly regulate mutated mRNA expression that contains repeated sequences such as, for example, 3 'or 5' CUG, CAG and / or CCUG. More specifically, it has been shown that antisense tricycle-DNA oligonucleotides are effective in facilitating exon hopping during pre-mRNA processing, in masking intronic silencer sequences and / or stem-loop sequences in pre-mRNA and in targeting of RNase-mediated destruction of mRNA.
[0004] [0004] Duchenne Muscular Dystrophy (DMD) is the most common hereditary myopathy, afflicting about one in 3,500 men regardless of ethnicity. The main consequence of DMD is that muscle fibers become particularly fragile and the natural activity of the muscle causes widespread damage to muscle tissue. The lack of dystrophin makes these muscle fibers particularly vulnerable to mechanical stress and undergo recurrent necrosis cycles. As a result, patients who exhibit progressive weakness of skeletal muscles, which are eventually replaced by adipofibrotic tissue, leading to loss of ambulation around the age of twelve, as a consequence premature death is caused either by respiratory failure or cardiomyopathy between the second and the fourth decade. In addition, about a third of DMD patients also exhibit cognitive impairment suggesting a noticeable disruption of neural and brain function. DMD affects all voluntary muscles and involves the cardiac and respiratory muscles in advanced stages of the disease. The heart and CNS should therefore preferably be targeted by any therapy implanted to treat or alleviate the symptoms of patients with DMD.
[0005] [0005] A new class of compounds was found to have improved efficacy compared to tricycle-DNA oliconucleotides. The present invention describes the synthesis, properties and uses of tricyclophosphorothioate nucleotides. SUMMARY OF THE INVENTION
[0006] [0006] The present inventors have surprisingly shown that nucleic acid molecules that comprise tricycle-phosphorothioate nucleotides are, in addition to their shared ability with tc-DNA molecules to be active in a wide range of muscles, highly effective in penetrating cardiac tissue and are highly active in cardiac cells. It has also been shown that such tricyclo-phosphorothioate nucleotides are able to rescue the expression of a protein, particularly dystrophin, in the CNS after systemic delivery. The inventors have therefore shown the unexpected property of nucleic acid molecules comprising tricyclic phosphorothioate nucleotides to cross the blood-brain barrier.
[0007] [0007] The invention, therefore, relates to nucleic acid molecules comprising tricyclonucleosides joined by internucleoside phosphorothioate bonds (3'-OPS-0-5 'bonds). The nucleic acid molecules of the invention are also referred to as "tricycle-phosphorothioate DNA" or "tc-DNA-PS" in the present disclosure.
[0008] [0008] The invention also relates to a composition comprising a tc-DNA-PS and a carrier. The composition can, in particular, be a pharmaceutical composition, wherein the carrier is a pharmaceutically acceptable carrier. The composition of the invention may also optionally comprise an additional active agent.
[0009] [0009] The present invention also relates to a method for synthesizing tc-DNA-PS molecules.
[0010] [0010] The invented nucleic acid molecules are particularly useful as antisense oligonucleotides (AONs), particularly for obtaining an antisense effect on muscles and cardiac cells or on the CNS, particularly after the systemic delivery of AON. The present invention, therefore, also provides tc-DNA-PS AONs.
[0011] [0011] As the inventors showed that, after the systemic delivery, an AON of tc-DNA-PS according to the invention can correct the expression of dystrophin in the muscles, in the cardiac tissue and in the CNS, the invention also refers to methods that employ tc-DNA-PS AONs to treat diseases. Representative diseases include, for example, heart disease, such as hypertrophic obstructive cardiomyopathy caused by cMYBP-C mutations and neuromuscular diseases such as Duchenne Muscular Dystrophy, Spinal Muscular Atrophy and Steinert's Myotonic Dystrophy. More generally, the invention relates to a method for correcting the abnormal gene expression in a cell of an individual, wherein the method comprises administering to the individual an antisense tc-DNA-PS oligonucleotide, wherein said oligonucleotide tc-DNA-PS antisense is complementary to a portion of RNA encoded by said gene. In a preferred embodiment, said tc-DNA-PS antisense oligonucleotide is administered peripherally to the individual in an amount sufficient to correct said abnormal expression. Preferred peripheral administration includes systemic injection, such as intravenous, subcutaneous, intraperitoneal or intraarterial injection.
[0012] [0012] The invention also relates to a method for treating a genetic disease caused by abnormal gene expression in an individual's cell, wherein the method comprises administering to the individual an antisense tc-DNA-PS oligonucleotide, in which the said tc-DNA-PS antisense oligonucleotide is complementary to a portion of an RNA encoded by said gene. Said tc-DNA-PS antisense oligonucleotide is preferably administered peripherally to the individual in an amount sufficient to correct said abnormal expression. In particular, the tissue or cell can be selected from CNS, cardiac and muscle cells or tissues.
[0013] [0013] Tc-DNA-PS in the present invention are shown to be transported in the bloodstream after systemic intravenous / intraperitoneal or subcutaneous application to all skeletal muscles, CNS and cardiac muscle and being taken by these tissues.
[0014] [0014] Other objectives and applications will become apparent from the following detailed description of the invention. DESCRIPTION OF THE DRAWINGS
[0015] [0015] Figure 1 shows the chemical structures and sequences of morpholine PMO, 2'0-Me-PS-RNA, Tc-DNA and Tc-phosphorothioate DNA oligonucleotides used for exon 23 pre-mRNA dystrophin hop in mouse mdx.
[0016] [0016] Figure 2 is a schematic representation of the mdx mutation and splice switching logic for dystrophin rescue. The mdx mutation consists of a single base change (transition from C to T) in exon 23 of the dystrophin gene (A). Such a transition generates a premature termination codon (UAA) that abolishes dystrophin synthesis (B). According to the exon phasing around exon 23, it is possible to skip the exon that houses the premature termination codon during pre-mRNA splicing using the main antisense oligonucleotide ring motifs involved in the definition of exon 23 ( Ç). Exon 23 without the resulting mRNA can be translated into a truncated but still functional dystrophin.
[0017] [0017] Figure 3 shows rescue of dystrophin diffused in mdx muscles after systemic delivery of tc-DNA-PS (M23D + 2-13).
[0018] [0018] Detection of dystrophin mRNA with exon 23 jump in mdx muscles after systemic delivery of tc-DNA-PS (Dose: 200 or 50 mg / kg body weight / twice a week; Route: intravenous; Duration: 12 weeks). RNA samples were analyzed 2 weeks after the end of the treatment by nested RT-PCR as previously described. The 688 bp fragment that corresponds to the exon 23 mRNA was detected in all muscles tested including the heart. A diagram is also depicted showing different exons present in the dystrophin pre-mRNA (with or without exon 23 jump) and the position in exons 20 and 26 of the primers used for nested PCR. (B) Western transfer of total protein (50 μg) extracted from different muscles of the treated mice, stained with NCL-DYS1. Arrows indicate full-length dystrophin as detected in samples of normal muscle used for comparison. It is noted that the 8 kD difference between wild and rescued proteins could not be resolved in this type of gel.
[0019] [0019] Percent of exon jump analyzed by Taqman's qPCR (left panel) and percentage of rescued dystrophin evaluated by Western blot transfer (right panel). The exon 23 jump is expressed as a percentage of total dystrophin, measured by the exon 4-5 expression level, after normalization with an endogenous control. To quantify the levels of dystrophin protein restored in various muscles, membranes were converted into numerical prints by scanning and the band intensities were analyzed using the ImageJ 1.46r software. Dystrophin levels are expressed as a percentage compared to levels in wild type tissue. The analysis involved 3 animals per group.
[0020] [0020] Figure 4 is an immunostaining of dystrophin from normal (A), untreated mdx muscles (B) and mouse muscles 12 weeks after intravenous injections of tc-PS (+2 -13) (C) gastrocnemius oligonucleotide, (D) anterior tibial, (E) diaphragm. (F and G) show dystrophin staining in the wild and treated mdx heart, respectively. The nuclei were counterstained with Dapi. Legend: Detection of dystrophin in mdx muscles after systemic treatment with the tc-DNA-PS M23D oligomer (+ 2-13). Mdx mice were treated weekly with intravenous injections of M23D (+ 2-13) for 12 weeks, at a dose of 200 mg / kg of body weight. Two weeks after the last injection, the muscles were dissected and processed for immunofluorescence analysis involving staining with the monoclonal dystrophin antibody NCL-DYS2. (A and B) shows cross sections of normal and mdx muscles. (C, D and E) show immunological identification of dystrophin from treated mdx muscle samples: gastrocnemius, anterior tibial and diaphragm, respectively. (F and G) show dystrophin staining in normal heart and treated mdx, respectively.
[0021] [0021] Figure 5 presents an experiment on comparing intravenous versus subcutaneous delivery of tc-PS oligonucleotide.
[0022] [0022] The mdx animals were treated for 8 weeks with 100 mg / kg of tc-DNA-PS (M23D + 2-13) twice a week delivered by injections or intravenous (A) or subcutaneous (B). Both routes of administration generate similar results as shown by Western blot transfer analysis (each row was loaded with 100 μg of total protein) from muscle samples stained with the monoclonal antibody NCL-DYS1. The analysis was performed 2 weeks after the end of treatment. Arrows indicate full-length dystrophin as detected from the row loaded with wild-type muscle.
[0023] [0023] Figure 6 shows photographs of western blot transfers that show the expression of dystrophin protein in cardiac muscle in mdx mice after systemic treatment with tc-DNA-PS M23D oligomer (+ 2-13).
[0024] [0024] Results of Western blot transfer analysis (using the monoclonal antibody NCL-DYS1 of dystrophin) of total protein extracts (100 μg loaded) isolated from the hearts of 3 mdx mice treated with the tc-DNA oligomer M23D (+ 2-13) (white stars) (biweekly injections - subcutaneous and intravenous - at 100 mg / kg for 8 weeks) (A); and 3 mdx mice treated under the same conditions with the tc-DNA-phosphorothioate oligomer (-PS) M23D (+ 2-13) (black stars) (B). The arrow indicates the 427 kD full-length dystrophin, as detected in the corresponding wild-type control rows for semi-quantitative comparison of dystrophin signal detection. Wild type heart extracts were diluted 30 to 5% for (A) and 10 to 1.25% for (B) in mdx heart extracts in order to normalize the amount of protein loading to 100 μg per row.
[0025] [0025] Figure 7 is an agarose gel from nested PCR reactions that shows the dystrophin pre-mRNA hop in the CNS of mdx mice treated with either the tc-DNA M23D (+ 2-13) oligonucleotide or the tc oligonucleotide -DNA-PS M23D (+ 2-13). The injections were performed either systemically or by means of stereotaxic injection in the Cisterna Magna.
[0026] [0026] Caption: Detection of dystrophin mRNA with exon 23 jump in the central nervous system of mdx after systemic treatment with either oligomers or tc-DNA or tc-DNA-PS M23D (+ 2-13). The mdx mice were treated biweekly with subcutaneous and intravenous injections of M23D (+ 2-13) (main chains of tc-DNA or tc-DNA-PS) for 8 weeks, with a dose of 100 mg / kg of body weight. One week after the last injection, the brains were dissected and processed for detection of dystrophin mRNA with exon hop 23. RNA samples were analyzed by nested RT-PCR using primers (Fo (off) / Fi ringing exon) (inside) 20 and Ro / Ri 26 ringing exon and junction 22 to 24, respectively) allowing specific recognition of the skipped messenger as a 398 bp fragment. It is also noted that since the Ri primer specifically annexes the boundary between exon 22 and exon 24, dystrophin mRNA without leaping is not amplified and the 398 bp band can only be detected in samples containing dystrophin mRNAs without exon 23. SM, size markers; row 2 - untreated mdx cerebellum; rows 3 to 5 - cortex, hippocampus and cerebellum in CNS of mdx one month after a stereotactic injection of 400 μg of tc-DNA M23D (+ 2-13) in Cisterna Magna; rows 6 to 8 - cerebellum in 3 mdx mice after systemic treatment with tc-DNA M23D (+ 2-13) (white *); rows 9 to 11 - cerebellum in 3 mdx mice after systemic treatment with tc-DNA phosphorothioate (-PS) M23D (+ 2-13) (black *). Detection of exon 23 leaps in cortex, hippocampus and cerebellum after 5 weeks of systemic treatment using a dose of only 25 mg / kg / week of tc-DNA-PS M23D (+ 2-13). It is noted that the systemic treatment with the tc-DNA-PS M23D oligomer (+ 2-13) rescues the dystrophin mRNA in CNS after systemic administration, whereas the tc-DNA form requires intracerebral delivery.
[0027] [0027] Figure 8 shows rescue of dystrophin mRNA in the CNS of mdx mice after systemic delivery of tc-DNA-PS (M23D + 2-13) in two different dosages.
[0028] [0028] Effects of tc-DNA-PS M23D on CNS after systemic delivery (Dose: 200 and 50 mg / kg / week; Route: intravenous; Duration: 12 weeks). RNA or whole brain or cerebellum samples were analyzed by nested RT-PCR using specific primers that specifically amplify dystrophin mRNA with exon 23 hop (398 bp amplicon). A sample of the anterior tibial of a treated mdx was used as a positive control. (B) Percent of exon leap analyzed by Taqman qPCR in the whole brain and the cerebellum of treated animals (n = 3 per group).
[0029] [0029] Figure 9 presents experiments that show that the systemic delivery of tc-DNA-PS (M23D + 2-13) improved the mdx phenotype. (A) Levels of serum creatine kinase in treated animals compared to wild-type and untreated mdx (n = 3 per group). P <0.001 for 200mg / kg / week and p <0.05 for 50mg / kg / week. (B) For both regimens (200 or 50 mg / kg), serum ALT and AST levels show that tc-DNA-PS did not produce liver toxicity. P> 0.05 compared to untreated mdx mice. (C) Improvement of muscle function in mice treated with tc-DNA-PS. The anterior tibial muscles (TA) of treated mdx mice were analyzed for their specific strength (maximum normalized strength for cross-sectional area). p <0.001 for 200 mg / kg / week and p <0.05 compared to untreated mice. (D) The percentage of strength drop was assessed by measuring the strength deficit after a series of three eccentric concentrations. The values confirm that the muscles in treated mdx animals were more resilient than in untreated mdx. The strength drop in treated mice (200 and 50mg / kg / week) is not significantly different from the wild type.
[0030] [0030] Figure 10 shows experiments showing dystrophin rescue in the dKO mouse model after the intravenous delivery of tc-DNA-PS (M23 + 2-13).
[0031] [0031] Systemic treatment with tc-DNA-PS (M23D + 2-13) (Dose: 200 mg / kg / week; Via: alternating intravenous and subcutaneous) prevents the onset of dystrophic pathology in dKO mice. Photograph of an untreated dKO mouse at 12 weeks of age (left), showing strong kyphosis and joint contractures compared to a treated litter (right), which looks healthy. Treatment was started at 3 weeks of life. (A) Detection of dystrophin mRNA with exon 23 jump in dKO muscles after systemic delivery of tc-DNA-PS (Dose: 200 mg / kg / week; Route: alternating intravenous and subcutaneous; Duration: 20 weeks). RNA samples were analyzed 2 weeks after the end of the treatment by nested RT-PCR as previously described. The 688 bp fragment that corresponds to the exon 23 mRNA was detected in all muscles tested including the heart. (C - F) Dystrophin immunostaining in cross sections of treated dKO muscles: (C) anterior tibial; (D) gastrocnemius; (E) diaphragm; (F) heart. The sections of untreated animals were devoid of dystrophin staining. The nuclei were stained with Dapi (blue). (G) Percent of exon jump analyzed by Taqman qPCR in different muscles and brain after 5, 11, 18 or 20 injections.
[0032] [0032] Figure 11 is a diagram showing the expected results after one year treatment. As a result of the cumulative effect of repeated systemic injections, it is expected that the maximum treatment effect can be achieved in about 20 weeks from the diaphragm (observed during the course of the experiment) and 40 weeks for other skeletal muscles. It would also be expected that the exon jump could reach 60% in cardiac muscle and about 15% in CNS.
[0033] [0033] Figure 12 shows experiments that show that the systemic delivery of tc-DNA-PS (M23D + 2-13) improved the dKO phenotype.
[0034] [0034] Levels of serum creatine kinase in treated animals compared to wild-type and untreated dKO (n = 5 per cohort) (p <0.05). (B and C) Mice were analyzed at 10 weeks of age with cages of open field behavioral activity. (B) cumulative active time and (C) distance covered for 1 hour (n = 5 per cohort). (D - F) Improvement of muscle function in mice treated with tc-DNA-PS. (D) Evaluation of muscle function of the forelimb shows physical improvement in treated dKO P <0.05. (E) Long finger extensor muscles (EDL) of treated dKO mice were analyzed for their specific strength (maximum normalized strength for cross-sectional area). (F) The percentage of strength drop was assessed by measuring the strength deficit after a series of 5 eccentric concentrations. p <0.05 compared to untreated dKO mice. The values confirm that the muscles in treated dKO animals were more resilient than in untreated dKO. Error bars are shown as mean ± SEM (N = 5 per cohort).
[0035] [0035] Figures 13 and 14 show experiments that show the lasting effect of tc-DNA-PS (M23D + 2-13). Figure 13 shows the pharmacokinetics of tc-DNA-PS (M23D + 3-13) after intravenous injection (A). The mice received a single injection of oligonucleotides at a concentration of 200 mg / kg. Serum samples were collected at different points in time and analyzed by HPLC-MS / MS to assess the levels of tc-DNA-PS in the blood compartment. (B) Treatment durability was assessed by comparing the percentage of jumping in animals loaded with the same amount of tc-DNA-PS (total amount of about 15 mg), but analyzed either at 2 weeks or 13 weeks after the end of the treatment. treatment. Importantly, about 3 months after the last injection, the jump levels were still significantly very high representing almost half of the initial result as measured at 2 weeks after treatment. This suggests that tc-DNA-PS are stable in cells and could be used again over time, thus limiting the need to fill tissues as would normally be required if these oligos were destroyed and titrated by their target mRNA.
[0036] [0036] Figure 14 shows another way to test the persistence of the treatment effect. Three sets of animals were treated with the same amount of tc-DNA-PS M23D (+ 2-13). Set 1 for 12 weeks at 200 mg / kg / week, Set 2 for 12 weeks at 50 mg / kg / week and Set 3 for 4 weeks at 200 mg / kg / week followed by 8 weeks at 50 mg / kg / week. The muscles were collected 2 weeks after the end of the treatment and analyzed by Taqman RT-qPCR. (n = 3 per cut).
[0037] [0037] Figure 15 shows the effect of systemic delivery of tc-DNA-PS (ISS7) in the SMA mouse model (FVB. Cg-Tg (SMN2) 2Hung Smn1tm1Hung / J).
[0038] [0038] SMA Type III mice (FVB. Cg-Tg (SMN2) 2Hung Smn1tm1Hung / j) are knocked out for Smn (Smn1 - / -) and contain an SMN2 transgene made of two tandem copies of the human SMN2 gene. These animals exhibit typical characteristics including necrosis of the tail departure in about a month of life. Such necrosis progressively extends to the outside of the ears and feet and later in life these animals show muscle weakness.
[0039] [0039] The photograph shows 3 type III individuals (one month old). The upper is an untreated control that shows typical necrosis of the tail; the other two, treated with tc-DNA-PS (ISS7) do not show such a characteristic that indicates SMN2 gene are rescued by inclusion of exon 7 thus generating SMN: They received a single ICV (intracerebroventricular) injection at birth (5 μl containing 20 μg of tc-DNA-PS (ISS7)) and repeated SC injections (subcutaneous) once a week at a dose of 200 mg / kg.
[0040] [0040] Figure 16 presents an experiment that shows the in vitro efficacy of a tc-DNA oligonucleotide that targets CUG amplifications.
[0041] [0041] Figure 17 presents an experiment that shows the in vitro efficacy of a tc-DNA oligonucleotide that targets CUG amplifications.
[0042] [0042] Figures 18 to 20 show an experiment that shows the in vitro efficacy of a tc-DNA oligonucleotide that targets CUG amplifications. DETAILED DESCRIPTION
[0043] [0043] The present invention is based on the unexpected revelation that DNA molecules of tricyclo-foforothioate, as exemplified by the antisense tc-DNA-PS (AON) oligonucleotide designated M23D (+ 02-13), can be delivered to cardiac cells and in the central nervous system (CNS) after intravenous administration to restore a mutated gene, such as a mutated dystrophin gene.
[0044] [0044] This revelation is quite surprising since the tricycle-DNA version of the oligonucleotide (ie, an oligonucleotide that comprises classic phosphodiester bonds between tricyclonucleosides) is not as efficient in modifying gene expression in cardiac cells or in the CNS after systemic administration. In addition, neither PMO nor 2'OMe-PS-RNA were effective in modifying gene expression in cardiac cells at doses acceptable for use in human subjects (Yokota, T et al Ann Neurol 2009; Mol Ther. June 2010; 18 (6): 1,210 to 7. Pre-clinical studies of PK and PD in antisense 2'-0-methyl phosphorothioate RNA oligonucleotides in the mdx mouse model. Heemskerk H, Winter C, van Kuik P, Heuvelmans N, Sabatelli P, Rimessi P, Braghetta P, van Ommen GJ, by Kimpe S, Ferlini A, Aartsma-Rus A, van Deutekom JC.). For these chemicals, getting into cardiac cells required either exceptionally high doses such as 3g / kg - 300 times the dose used in clinical trials today (Gene Ther. January 2010; 17 (l): 132 to 40. Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino.Wu B, Lu P, Benrashid E, Malik S, Ashar J, Doran TJ, Lu QL) or conjugated penetration peptides or mechanical stress such as ultrasound (Mol Ther. July 2011; 19 (7): 1.295 to 303. Pip5 transduction peptides target the exon hop, with high efficacy, of oligonucleotide-mediated dystrophin in the heart and phenotypic correction in mdx mice. Yin H, Saleh AF, Betts C, Camelliti P, Seow Y, Ashraf S, Arzumanov A, Hammond S, Merritt T, Gait MJ, Wood MJ; Ultrasound Med Biol. June 2009; 35 (6): 976 to 84. Microbubble stability is a primary determinant of gene transfer-mediated microbubble ultrasound efficacy Alter J, Senn oga CA, Lopes DM, Eckersley RJ, Wells DJ. )
[0045] [0045] The present disclosure will find wide application in the treatment of genetic diseases, in general, and more specifically, in the treatment of a neuromuscular or musculoskeletal disease, such as Duchenne Muscular Dystrophy, Spinal Muscular Atrophy and Myotonic Steinert Dystrophy, and in the treatment heart disease and CNS.
[0046] [0046] Definitions
[0047] [0047] As used herein, the term "phosphorothioate bond" refers to a chemical portion of 5 '... -0-P (S) -0 -... 3' between two adjacent nucleosides in a nucleic acid molecule.
[0048] [0048] As used herein, the term, “tricycle-DNAs (tc-DNA)” refers to a class of restricted DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict flexibility of the main chain and to optimize the main chain geometry of the torsion angle γ (Ittig et al, Nucleic Acids Res. 32: 346 to 353 (2004); Ittig et al, Prague, Academy of Sciences of the Czech Republic. 7 : 21 to 26 (Coll. Symp. Series, Hocec, M., 2005); Ivanova et al., Oligonucleotides 17: 54 to 65 (2007); Renneberg et al., Nucleic Acids Res. 30: 2. 751 to 2 757 (2002); Renneberg et al., Chembiochem. 5: 1. 114 to 1. 118 (2004); and Renneberg et al., JACS. 124: 5. 993 to 6. 002 (2002)). tc-DNAs containing adenine and homobasic thymine form extraordinarily stable A-T base pairs with complementary RNAs.
[0049] [0049] As used herein, the term "tricyclonucleoside" refers to a subunit of a nucleic acid molecule that has the following formula:
[0050] [0050] As used herein, the term "antisense oligonucleotide (AON)" refers to an oligonucleotide that can interact with and / or hybridize to a pre-mRNA or an mRNA that has a complementary nucleotide sequence, as well modifying gene expression.
[0051] [0051] As used herein, a "base" refers to typical DNA and RNA bases (uracil, thymine, adenine, guanine and cytosine) and modified bases or base analogues (eg, 5-methyl cytosine, 5-bromouracil or inosine). A base analog is a chemical whose molecular structure mimics that of a typical DNA or RNA base.
[0052] [0052] As used herein, "complementary" refers to a nucleic acid molecule that can form hydrogen bond (s) with another nucleic acid molecule or by traditional Watson-Crick base pairing or other types not traditional pairing methods (eg, Hoogsteen or inverted Hoogsteen hydrogen bonding) between complementary nucleotides or nucleosides. In reference to the tc-DNA-PS AON of the present disclosure, the free binding energy for a tc-DNA-PS AON with its complementary sequence is sufficient to allow the relevant function of the tc-DNA-PS AON to proceed and there is a sufficient degree of complementarity to avoid non-specific binding of the tc-DNA-PS AON to non-target sequences under conditions where specific binding is desired, that is, under physiological conditions in the case of ex vivo or in vivo therapeutic treatment. The determination of free binding energies for nucleic acid molecules is well known in the art (see, for example, Turner et ah, CSH Symp. Quant. Biol. £ 77: 123 to 133 (1987); Freier et ah, Proc. Nat. Acad. Sci. USA 83: 9,373 to 77 (1986); and Turner et al, J. Am. Chem. Soc. 109: 3,783 to 3,785 (1987)). Thus, "complementary" (or "specifically hybridizable") are terms that indicate a sufficient degree of complementarity or precise pairing so that a stable and specific bond occurs between a tc-DNA-PS AON and a pre-mRNA target or mRNA.
[0053] [0053] It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules can be less than fully complementary. Complementarity is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then the base match of 5, 6, 7, 8, 9 or 10 nucleotides between the first and second molecules of nucleic acid represents 50%, 60%, 70%, 80%, 90% and 100% complementarity, respectively. "Perfectly" or "fully" complementary nucleic acid molecules mean those in which all the contiguous residues of a first nucleic acid molecule will be hydrogen bonded with the same number of contiguous residues in a second nucleic acid molecule, where the molecules nucleic acid or both have the same number of nucleotides (that is, they are the same length) or the two molecules have different lengths.
[0054] [0054] As used herein, the terms "precursor mRNA" or "pre-mRNA" refer to a single immature strand of messenger ribonucleic acid (mRNA) that contains one or more intervention sequences (introns). The pre-mRNA is transcribed by an RNA polymerase from a DNA model in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Once a pre-mRNA has been completely processed by removing intron splicing and exon joining, it is referred to as "messenger RNA" or "mRNA," which is an RNA that is understood exclusively by exons. eukaryotic pre-mRNAs exist only transiently and are fully processed in mRNA. When a pre-mRNA has been properly processed into an mRNA sequence, it is exported outside the nucleus and eventually translated into a protein by ribosomes in the cytoplasm.
[0055] [0055] As used herein, the terms "splicing" and "processing" refer to the modification of a pre-mRNA after transcription, in which introns are removed and exons are joined. Splicing occurs in a series of reactions that are catalyzed by a large complex of RNA and protein composed of five small nuclear ribonucleoproteins (snRNPs) referred to as a spliceosome. Within an intron, a 3 'splice site, a 5' splice site and a branch site are required for splicing. The RNA components of snRNPs interact with the intron and may be involved in catalysis.
[0056] [0056] Pre-mRNA splicing involves two sequential biochemical reactions. Both reactions involve spliceosome transesterification between RNA nucleotides. In a first reaction, the 2'-OH of a specific branching point nucleotide within an intron, which is defined during spliceosome assembly, performs a nucleophilic attack on the first intron nucleotide of the 5 'splice site that forms a loop intermediate. In a second reaction, the 3'-OH of the released 5 'exon performs a nucleophilic attack on the last nucleon of the intron at the splice site 3' thus uniting the exons and releasing the intron loop. Pre-mRNA splicing is regulated by several factors such as sequences of exonic splice enhancers or inhibitors and particularly also by intronic silencer (ISS) sequences and terminal rod-loop (TSL) sequences.
[0057] [0057] As used herein, the terms "intronic silencer sequences (ISS)" and "terminal rod-loop (TSL)" refer to sequence elements within introns and exons, respectively, that control alternative splicing by binding trans-acting protein factors within a pre-mRNA, thus resulting in the differential use of splice sites. Typically, sequences of intronic mufflers are between 8 and 16 nucleotides and are less converted than splice sites to exon-intron junctions. Terminal stem-loop sequences are typically between 12 and 24 nucleotides and form a secondary loop structure due to complementarity and, therefore, binding, within the 12 to 24 nucleotide sequence.
[0058] [0058] By "individual" is meant an organism that is a donor or recipient of explanted cells or of the cells themselves. "Individual" also refers to an organism to which the nucleic acid molecules of this disclosure can be administered. In one embodiment, an individual is a mammal or mammalian cell. In another embodiment, an individual is a human or human cell.
[0059] [0059] As used herein, the term "therapeutically effective amount" means an amount of tc-DNA-PS molecule (e.g., an AON) that is sufficient in the individual (e.g., human) to which it is administered , to treat or prevent a specific disease, disorder or condition. The tc-DNA-PS molecule of the present invention, individually or in combination or in conjunction with other drugs, can be used to treat diseases or conditions, particularly those discussed in this document. For example, to treat a particular disease, disorder or condition, tc-DNA-PS can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under adequate conditions for treatment.
[0060] [0060] As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce a similar allergic or unpleasant reaction, such as gastric discomfort, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the European or American Pharmacopoeia or another pharmacopoeia generally known for use in animals and more particularly in humans.
[0061] [0061] As used herein, the term "isolated" means that the material referred to is removed from its native environment, for example, a cell. Thus, an isolated biological material may be free of some or all of the cellular components, that is, components of the cells in which the native material occurs naturally (for example, cytoplasmic or membrane complement).
[0062] [0062] The term "purified" as used in this document refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, that is, contaminants, including native materials from which the material is obtained. For example, a purified tc-DNA-PS molecule is preferably substantially free of cell or culture components, including tissue culture components, contaminants and the like. As used herein, the term "substantially free" is used operationally in the context of analytical testing of the material. Preferably, the purified material substantially free of contaminants is at least 50% pure; most preferably at least 90% pure and even more preferably at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay and other methods known in the art.
[0063] [0063] In the present description, any concentration range, percentage range, ratio range or whole number range should be understood including the value of any integer within the quoted range and, where appropriate, fractions thereof (such as a tenth and one hundredth of an integer), unless otherwise stated. In addition, any numerical range mentioned in this document related to any physical resource, such as polymeric subunits, size or thickness, should be understood to include any integer within the quoted range, unless otherwise indicated. As used herein, "about" or "consisting essentially of" means ± 20% of the indicated range, value or structure, unless otherwise stated.
[0064] [0064] As used herein, the terms "includes" and "comprises" are used interchangeably. It should be understood that the terms "one" and "one" as used in this document refer to "one or more" of the listed components. The use of the alternative (for example, "or") should be understood to mean either, both or any combination of the alternatives.
[0065] [0065] The term "about" or "approximately" means within a statistically significant range of a value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or track. The allowable variation included by the term "about" or "approximately" depends on the particular system under study and can be easily perceived by one skilled in the art.
[0066] [0066] In the nomenclature used in this document to designate AONs, as in M23D (+ 02-13), M means mouse, 23 is the exon id, D means donor site at the 3 'end of the exon, +2 indicates that the antisense starts inside the exon, 2 nucleotides before the D site, -13 indicates that the antisense ends at the 13th nucleotide of the downstream intron.
[0067] [0067] Tricyclic phosphorothioate DNA molecules of the invention and compositions containing the same
[0068] [0068] An object of the invention relates to a nucleic acid molecule comprising tricyclonucleosides joined by internucleoside phosphorothioate bonds (3'-OPS-0-5 'bonds), also referred to as "tricyclophosphorothioate DNA "or" tc-DNA-PS "in the present disclosure.
[0069] [0069] The nucleic acid molecule of the invention derives from the improvement of DNA chemistry containing tricyclonucleoside DNA, in which phosphodiester bonds are replaced by phosphorothioate bonds.
[0070] [0070] In accordance with the present disclosure, a nucleic acid of the invention comprises at least two adjacent tricyclonucleosides joined by a phosphorothioate bond. This sequence of chemical portions was never revealed before the present study. It should be understood that the nucleic acid molecule of the invention can also comprise nucleosides with different chemistry, such as nucleosides containing classic ribose and deoxyribose, LNA nucleosides and the like. The nucleic acid molecule of the invention may also contain other types of internucleoside bonds, in addition to the phosphorothioate bond, for example, classical phosphodiester bond. However, the invention preferably relates to nucleic acid molecules in which the proportion of tricyclonucleosides represents at least 50%, preferably at least 60%, 70%, 80%, 90% or 95% of the nucleosides totals in the nucleic acid molecule. In addition, the invention preferably relates to nucleic acid molecules in which the proportion of internucleoside phosphorothioate bonds represents at least 50%, preferably at least 60%, 70%, 80%, 90% or 95 % of total internucleoside bonds in the nucleic acid molecule. In a particular embodiment, all the nucleosides in the nucleic acid molecule of the invention are tricyclonucleosides. In another embodiment, all bonds between subunits are phosphorothioate bonds.
[0071] [0071] In a particularly preferred embodiment, the nucleic acid molecule of the invention is a tricyclo-phosphorothioate nucleic acid molecule comprising subunits of nucleosides joined by links between subunits, where all nucleosides are tricyclonucleosides and all links between subunits are phosphorothioate bonds.
[0072] [0072] The nucleoside subunits comprised in the nucleic acid of the invention can be selected to be in a defined sequence, such as a sequence of bases that can hybridize specifically to a single-stranded target nucleic acid sequence or a sequence that will allow the formation of a triple structure between the nucleic acid of the invention and a target nucleic acid duplex. The target nucleic acid sequences can be RNA and DNA sequences. When desirable, the nucleic acids of the present invention can be identified with a reporter group, such as radioactive identifications, biotin identifications, fluorescent identifications and the like, to facilitate the detection of the nucleic acid itself and its presence in, for example, hybridization complexes .
[0073] [0073] The size of the nucleic acid molecule of the invention will depend on the particular use for which it is prepared. For example, the tc-DNA-PS molecule of the invention can be at least 3 nucleotides in length, particularly at least 5, 10, 20, 30, 40 or 50 nucleotides in length. In a particular embodiment, the tc-DNA-PS molecule of the invention comprises between 3 and 50 nucleotides, particularly between 5 and 21 nucleotides, particularly between 6 and 18 nucleotides. Interestingly, oligonucleotides and tc-PS DNA can be reduced to 15 mer, while morpholino PMO and 2'O-Me-PS-RNA are usually transformed to 24 and 20 mer, respectively. Therefore, the invention particularly relates to tc-DNA-PS molecules that comprise, or that consist of, 15 nucleotides. In a further particular embodiment, the nucleic acid molecule of the invention comprises between 3 and 20 nucleotides, particularly between 10 and 15 nucleotides.
[0074] [0074] The synthesis of tricyclonucleosides is known in, for example, as described in Steffens, R. and Leumann, C. (1997) Nucleic-acid analogs with constraint conformational flexibility in the sugar-phosphate backbone "Tricyclo-DNA". Part 1. Preparation of [(5'R, 6'R) -2-deoxy-3 ', 5'-ethano-5', 6'-methano- -D-ribofuranosyl] thymine and -adenine, and the corresponding phosphoramidites for oligonucleotide synthesis. Helv. Chim. Ata, 80, 2,426 to 2,439 and in Renneberg, D. and Leumann, C. J. (2002) Watson-Crick base-pairing properties of tricyclo-DNA. J. Am. Chem. Soc, 124, 5,993 to 6,002.
[0075] [0075] The synthesis of phosphorothioate tc-DNA follows classic procedures in solid phase oligonucleotide synthesis according to the phosphoramidite approach (Oligonucleotide Synthesis - A Practical Approach, Oxford University Press, Oxford, 1984). In the synthesis method of the present invention, a first tricyclonucleoside is attached to a solid phase support (for example, to a porous glass controlled by long chain alkylamine (LCAA-CPG) by means of a succinyl linker). The first nucleotide additionally has a protected 5'-OH group (e.g., dimethoxytrityl group -DMT). The protected 5 'group is then deprotected to form a free 5'-OH group to which a second nucleotide is added. The free 5'-OH group of the first nucleotide is reacted with a 5'-protected, 3'-O-cyanoethyl-N, N-diisopropylaminophosphoramidite tricyclonucleoside. The internucleoside phosphoramidite group is then sulphorized to form a phosphorothioate internucleoside bond between the first and the second tricyclonucleosides. The unreacted 5'-OH groups of the first nucleotide are esterified (capped) to prevent failure sequence synthesis. That sequence is then repeated to add an additional tc-PS nucleotide as many times as necessary to form the complete desired nucleic acid sequence.
[0076] [0076] A particular embodiment of the method of synthesizing a nucleic acid according to the invention is described below, with reference to scheme 1.
[0077] [0077] Scheme 1: General protocol for the synthesis of tricycle-phosphorothioate-DNA (tc-DNA-PS)
[0078] [0078] The synthesis cycle in which an additional unit is linked to the growing chain consists of four sequential steps (a to d). After chain assembly, the oligonucleotide is separated from the solid support and unprotected in the usual way (NH3 cone, 55 ° C, 16 hours). The long chain alkylamine-controlled porous glass (LCAA-CPG), to which the first tricyclonucleoside is attached by means of a succinyl binder, is used as a solid support. The syntheses were generally performed on the scale of 1, 3 or 10 μmol in a Pharmacia gene assembler plus DNA synthesizer. Tricyclo-phosphorothioate-oligonucleotides are synthesized with a 5 'terminal phosphate or thiophosphate group to ensure the chemical stability of the 5' end (R. Steffens and CJ Leumann, J. Am. Chem. Soc, 1999, 121, 3. 249 to 3. 255). The conditions for each step a) to d) are given below and are optimized by a 10 μmol synthesis. a) Detritilation:
[0079] [0079] Cleaning with 3% dichloroacetic acid in 1,2-dichloroethane (DCE) for 1.5 min. Then wash with DCE and CH3CN.
[0080] [0080] b) Coupling:
[0081] [0081] The phosphoramidite solution (0.1 μΜ in CH3CN, 400 μl) and 5-ethylthietrazole activator (ETT, 0.25 M in CH3CN, 600μl) is applied to the solid support. Docking time: 9 minutes. Then wash with CH3CN.
[0082] [0082] CH3CN is used to build tc-T, tc-G and tc-C blocks. For reasons of solubility, the tc-A block construction is used in dry DCE as a solvent.
[0083] [0083] Sulfurization:
[0084] [0084] Bis (phenylacetyl) disulfide (PADS) in dry pyridine / CH3CN 1/1 (0, 2 M) is applied on the solid support for 3 minutes. Then wash with CH3CN.
[0085] [0085] d) Capping:
[0086] [0086] Untreated 5'-hydroxyl groups are capped using CapA solution (4-dimethylaminopyridine (DMAP, 0.5 M) in CH3CN) and CapB (acetic anhydride (AC20), collidine in CH3CN (2: 3 : 5)) for 20 seconds each. Then wash with CH3CN.
[0087] [0087] The tc-DNA phosphoramidite building blocks used for synthesis of the nucleic acid molecule of the invention can be synthesized as described in Steffens and Leumann, C. Helv. Chim. Minutes 80: 2,426 to 2,439 (1997). The chain extension cycles can be essentially identical to those for synthesis of natural oligodeoxynucleotide. See, Pharmacia LKB User's Manual (56-1111 -56) (Gene Assembler Special / 4 Primers).
[0088] [0088] The tc-DNA-PS molecule of the invention can be an antisense oligonucleotide complementary to a portion of an RNA encoded by a gene, particularly a human gene. The present invention thus also relates to an antisense tricyclophosphorothioate DNA oligonucleotide.
[0089] [0089] The tc-DNA-PS (or antisense oligonucleotide) molecule of the invention can be designed particularly:
[0090] [0090] - to perform exon skipping, particularly to skip one or more exons in a dystrophin gene;
[0091] [0091] - to facilitate the inclusion of an exon during the processing of a target pre-mRNA, particularly to facilitate the inclusion of exon 7 during the processing of an SMN2 pre-mRNA;
[0092] [0092] - to target a mutated mRNA that comprises excess CUG amplifications to prevent sequestration of nuclear proteins to expanded CUG repeats, for example, to target a mutated DM1 mRNA that comprises excess CUG amplifications;
[0093] [0093] - to facilitate the destruction of a mutated mRNA that comprises excess CUG amplifications, for example, facilitating the destruction of a mutated DM1 mRNA that comprises excess CUG amplifications.
[0094] [0094] The Tc-DNA-PS molecules of the present invention can be formulated into a composition with a carrier. The composition can be a pharmaceutical composition, with a carrier being a pharmaceutically acceptable carrier.
[0095] [0095] Thus, the invention also relates to a pharmaceutical composition comprising a nucleic acid of the invention, which is particularly an antisense oligonucleotide complementary to a portion of an RNA encoded by a gene, particularly a human gene and wherein said composition further comprises a pharmaceutically acceptable carrier. In addition, the invention also relates to a nucleic acid molecule of the invention, in combination with another therapeutic agent. The nucleic acid molecule of the invention and the other therapeutic agent can be formulated in a pharmaceutical composition or are part of a combined preparation (kit of parts), for simultaneous, separate or sequential use. The person skilled in the art will adapt the other therapeutic agent and the nucleic acid sequence of the invention to the particular disease being treated.
[0096] [0096] The tc-DNA-PS molecules described in this document can be mixed by addition with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; dispersing or humidifying agents can be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with aliphatic alcohols of long-chain, for example, heptadecaethyleneoxyethanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as sorbitol polyoxyethylene monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example, n-propyl ethyl or p-hydroxybenzoate. Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the active ingredient in admixture by addition with a dispersing or wetting agent, suspending agent and one or more preservatives.
[0097] [0097] According to a particular embodiment, the invention relates to a composition comprising a tc-DNA-PS molecule as described above and a pharmaceutically acceptable carrier, wherein the composition is an injectable composition. The tc-DNA-PS compositions can be in the form of a sterile injectable aqueous or oil suspension. The suspensions can be formulated according to the known technique using those dispersing and wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable solvent or diluent, for example, as a 1,3-butanediol solution. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils can conventionally be used as a solvent or suspending medium. For this purpose, any bland fixed oil can be used including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, find use in the preparation of injectables.
[0098] [0098] The present disclosure also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired tc-DNA-PS molecule of the invention in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co., A. R. Gennaro edit., 1985). For example, preservatives and stabilizers can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Additionally, antioxidants and suspending agent can be used.
[0099] [0099] The present disclosure also provides compositions and methods to facilitate the exon jump or to mask the intronic silencing or rod-loops ending in a pre-mRNA to target the destruction of mRNA in a cell or organism. In related embodiments, this disclosure provides methods and compositions that comprise a tc-DNA-PS molecule according to the invention to treat an individual, including a human cell, tissue or individual, who has a disease or is at risk of developing a disease , particularly one of the specific diseases as described in this document. In one embodiment, the method includes administering a tc-DNA-PS molecule of the present invention or a pharmaceutical composition containing the tc-DNA-PS molecule to a cell or an organism, such as a mammal, so that processing of a pre-mRNA to be modified and the destruction of a mRNA to be targeted. Mammalian individuals receptive to treatment using the compositions and methods of the present invention include those suffering from one or more disorders that are receptive to such treatment such as, Duchenne Muscular Dystrophy, Spinal Muscular Atrophy or Steinert Myotonic Dystrophy.
[0100] The tc-DNA-PS compositions of the present disclosure can be effectively employed as pharmaceutically acceptable formulations. Pharmaceutically acceptable formulations prevent, alter the occurrence or severity of, or treat (alleviate one or more symptoms to a detectable or measurable extent) a disease state or other adverse condition in a patient. A pharmaceutically acceptable formulation includes salts of the above compounds, for example, acid addition salts such as hydrochloric acid, hydrobromic acid, acetic acid and benzene sulfonic acid salts. A pharmaceutical composition or formulation refers to a composition or formulation in a form suitable for administration, for example, systemic administration, to a cell or patient such as a human. The appropriate forms, in part, depend on the use or the route of entry, for example, transdermal or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the tc-DNA-PS molecule is desirable for delivery). For example, pharmaceutical compositions injected into the bloodstream must be soluble. Other factors are known in the art and include considerations such as toxicity and forms that prevent the composition or formulation from having its effect.
[0101] [00101] The pharmaceutical compositions of this disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures thereof. Suitable emulsifying agents can be naturally occurring gums, for example, acacia gum or tragacanth gum; naturally occurring phosphatides, for example, soybeans, lecithin; or esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monoleate and condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monoleate.
[0102] [00102] The tc-DNA-PS molecule of this disclosure can be administered to a patient by any standard means, with or without stabilizers, buffers or the like to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for forming liposomes can be followed. Thus, nucleic acid molecules of the present disclosure can be administered in any form, for example, transdermally or by local, oral, rectal, intramuscular, intracardiac, intraperitoneal, locoregional, systemic injection (for example, intravenously or intra-arterial) or intrathecal.
[0103] [00103] This disclosure also features the use of compositions comprising surface-modified liposomes containing poly (ethylene glycol) lipids (modified PEG or long-circulating liposomes or reserve liposomes). Such formulations offer a method for increasing the accumulation of the tc-DNA-PS molecule of the invention in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thus enabling long blood circulation times and enhanced tissue exposure for the encapsulated tc-DNA-PS molecule (Lasic et al., Chem Rev. 95: 2. 601 to 2. 627 (1995) and Ishiwata et al, Chem. Pharm. Bull. 43: 1.005 to 1.011 (1995). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of nucleic acids, particularly in comparison to conventional cationic liposomes that are known to accumulate in MPS tissues (Liu et al, J. Biol. Chem. 42: 24. 864 to 24. 870 (1995); Choi et al, PCT Publication No. WO 96/10391; Ansell et al, PCT Publication No. WO 96/10390; Holland et al, PCT Publication No. WO 96/10392). Long-circulating liposomes are also possible protectors of tc-DNA- PS of the invention against nuclease degradation to a greater extent compared to cationic liposomes, based on their ca ability to avoid accumulation in metabolically aggressive MPS tissues such as liver and spleen.
[0104] [00104] A pharmaceutically effective dose is the dose required to prevent, inhibit the occurrence or treat (alleviate a symptom to some extent, preferably all symptoms) from an unhealthy state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal to be treated, the physical characteristics of the specific mammal under consideration, the concomitant medication and other factors than the elements skilled in medical techniques will recognize. For example, an amount between 0.1 mg / kg and 100 mg / kg of body weight / day of active ingredients is administered depending on the potency of the tc-DNA-PS molecule of this disclosure.
[0105] Dosage levels on the order of about 0.1 mg to about 140 mg per kilogram of body weight per week are useful in treating the conditions indicated in the present invention (about 0.5 mg to about 7 g per patient per week). The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the treated host and the particular mode of administration. Unit dosage forms generally contain from about 1 mg to about 500 mg of an active ingredient.
[0106] [00106] It should be understood that the specific dose level for any particular patient depends on a variety of factors including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration and rate of excretion, combination of drug and the severity of the particular disease undergoing therapy. After administering compositions according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% to about 99% reduction in one or more symptoms associated with the disease or disorder being treated, compared to treated with placebo or other suitable control subjects.
[0107] [00107] The tc-DNA-PS molecule of the invention can be administered to cells by a variety of methods known to those skilled in the art, including administration within formulations comprising the tc-DNA-PS molecule alone or comprising additionally one or more additional components, such as a carrier, diluent, excipient, adjuvant, emulsifier, buffer, stabilizer, pharmaceutically acceptable preservative or the like. In certain embodiments, the tc-DNA-PS molecule of the invention can be encapsulated in liposomes, administered by iontophoresis or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres or proteinaceous vectors (see, for example, publication PCT No. WO 00/53722).
[0108] [00108] The direct injection of the tc-DNA-PS molecule of this disclosure, whether intravenous, subcutaneous, intramuscular or intradermal, can occur using standard syringe and needle methodologies or by needle-free technologies, such as those described in Corny et al, Clin. Cancer Res. 5: 2,330 to 2,337 (1999) and PCT publication No. WO 99/31262.
[0109] [00109] Additional methods for delivering nucleic acid molecules are described, for example, in Boado et al, J. Pharm. Sci. 87: 1. 308 to 1. 315 (1998); Tyler et al, FEBS Lett. 421: 280 to 284 (1999); Pardridge et al, Proc. Nat'l Acad. Sci. USA 92: 5,592 to 5,596 (1995); Boado, Adv. Drug Delivery Rev. 15: 73 to 107 (1995); Aldrian-Herrada et al, Nucleic Acids Res. 26: 4,910 to 4,916 (1998); Tyler et al, Proc. Nat'l Acad. Sci. USA 96: 7,053 to 7,058 (1999); Akhtar et al, Trends Cell Bio. 2: 139 (1992); "Delivery Strategies for Antisense Oligonucleotide Therapeutics,” (ed. Akhtar, 1995); Maurer et al, Mol. Membr. Biol. 16: 129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol 137: 165-192 (1999); and Lee et al, ACS Symp. Ser. 752: 184-192 (2000) .These protocols can be used to supplement or complement virtually the delivery of any tc-DNA-PS molecule contemplated in this disclosure.
[0110] [00110] Treatment Methods
[0111] [00111] As mentioned above, the nucleic acid molecule of the present invention can be an antisense oligonucleotide (AON) designed to complement a specific mRNA or pre-mRNA. The antisense oligonucleotides of the invention can be used for the treatment of various diseases, countless of which are described below. Obviously, the illustrative diseases provided below do not limit the invention and the new chemistry provided in the present invention can be used to treat any disease that the versed element should be treatable by administering an AON.
[0112] [00112] Antisense Tricycle-phosphorothioate Oligonucleotides for the Treatment of Duchenne Muscular Dystrophy
[0113] [00113] Eat certain modalities, the present disclosure provides AONs that can be properly used for the treatment of Duchenne Muscular Dystrophy (DMD), a form linked to severe recessive muscular dystrophy that is characterized by the rapid progression of muscle degeneration, which leads eventually to loss in locomotion, paralysis and death. DMD is caused by a mutation, such as a non-sense or frame-shift mutation, within the dystrophin gene, which is located on the human X chromosome. The dystrophin gene encodes the dystrophin protein, an important structural component within muscle tissue that provides structural stability for muscle fiber sarcolemma as well as for the dystroglycan complex (DGC), located on the cell membrane. A non-sense or frame-shift mutation can result in premature translation termination and, therefore, a C-terminal truncated non-functional dystrophin protein.
[0114] [00114] DMD caused by one or more stop mutations or frame-shift mutations can be alleviated by excising one or more exons in order to restore the translational reading phase and, thereby, restore the mRNA sequence downstream of the mutation. To achieve this, as part of the present disclosure, the nucleic acid molecules according to the invention have been developed as antisense AONs for target regions within the pre-mRNA that can mask the spliceosomal recognition of one or more exon (s). By targeting these regions with tc-DNA-PS AONs, exons can be removed by splicing to produce mature, internally and partially deleted, but functional dystrophin mRNA.
[0115] [00115] Thus, the tc-DNA-PS AON described in the present invention is effective in facilitating the hop of one or more exons mutated in a dystrophin gene during the processing of a dystrophin pre-mRNA, thereby restoring the appropriate reading phase of the resulting dystrophin mRNA, which, when translated, produces a semifunctional dystrophin protein. Thus, the tc-DNA-PS AON disclosed in the present invention can be used therapeutically for patients affected by DMD.
[0116] [00116] As used herein, the term "exon hop" refers to the modification of pre-mRNA splicing by targeting splice donor and / or acceptor sites within a pre-mRNA with one or more complementary antisense oligonucleotides (AONs). By blocking the access of a spliceosome to one or more donor or splice acceptor sites or, in fact, any other site within an exon or intron involved in the definition of splicing, an AON can prevent a splicing reaction by causing, through addition, the deletion of one or more exons from a fully processed mRNA. The exon jump is achieved in the nucleus during the maturation process of pre-mRNAs. This includes the masking of important sequences involved in the splicing of targeted exons through the use of antisense oligonucleotides (AON) that are, for example, complementary to splice donor sequences within a pre-mRNA. The tc-DNA-PS AON provided in the present invention can be suitably employed for exon hopping by masking splice sites at intron / exon junctions within a dystrophin pre-mRNA thereby facilitating the deletion of a mutant exon during pre-mRNA processing for a mature mRNA.
[0117] [00117] For example, a non-sense or frame-shift mutation within exon 23 or exon 50 of a dystrophin gene produces a non-functional dystrophin protein truncated with a carboxy terminus. Through hybridization to nucleotides comprising a dystrophin pre-mRNA splice donor site at intron 23 or intron 51, respectively, and 5 'nucleotides adjacent to exon 23 or exon 51, the tc-DNA-PS AON revealed in the present invention is able to prevent the inclusion of exon 23 or mutated exon 51 in the mature mRNA transcript. The expression of this mature mRNA transcript produces a semifunctional dystrophin protein that is deleted from the amino acids encoded by exon 23 or exons 50 and 51, but which includes the dystrophin amino acids in both N-terminal and C-terminal for those deleted amino acids .
[0118] [00118] The tc-DNA-PS AON disclosed in the present invention for hopping an exon during processing of a dystrophin pre-mRNA typically contains between 6 to 22 contiguous tricycle-OS nucleotides, particularly between 8 to 20 nucleotides tricycle-PS, more particularly, between 10 and 18 contiguous tricycle-OS nucleotides, where 6 to 16 nucleotides, particularly 8 to 16 nucleotides of the tc-DNA-PS AON are complementary to an intron splice donor site of dystrophin pre-mRNA, where 2 to 8 nucleotides of the AON of tc-DNA-PS AON are complementary to a dystrophin pre-mRNA exon region and where the intron splice donor site is contiguous with e 5 'for the exon region. Depending on the precise application contemplated, the AON of tc-DNA-PS can be between 12 and 16 nucleotides or between 13 and 15 nucleotides and can comprise between 6 and 14 nucleotides that are complementary to the intron splice donor site and between 2 and 5 nucleotides that are complementary to the exon region.
[0119] [00119] Tc-DNA-PS AONs designed to skip a mutated exon 23 within a dystrophin pre-mRNA are exemplified in the present invention. The tc-DNA AON comprises the nucleotide sequence 5'-AACCTCGGCTTACCT-3 '(M23D (+ 02-13), SEQ ID NO: 1) and specifically hybridizes to nucleotides at the 3' end of pre-mRNA intron 23 dystrophin and for nucleotides at the contiguous 5 'end of exon 23 of dystrophin pre-mRNA. An alternative AON that can be used is the sequence 5 * -GGCCAAACCTCGGCTTACCT-3 * (M23D (+2 -18), SEQ ID NO: 2).
[0120] [00120] Also available are tc-DNA-PS AON designed to skip a 51 mutated exon within a dystrophin pre-mRNA. The tc-DNA AONs comprise a nucleotide sequence selected from the group consisting of 5'-AGAAATGCCATCTTC-3 '(H51 (+ 68 + 82), SEQ ID NO: 3), 5'-AAATGCCATCTTCCT-3' ( H51 (+ 70 + 84), SEQ ID NO: 4), 5'-TGCCATCTTCCTTGA-3 '(H51 (+ 73 + 87), SEQ ID NO: 5) and 5'-GCAGTTTCCTTAGTAA-3' (H51 (+40 +55), SEQ ID NO: 6) and specifically hybridizes to the nucleotides at the 3 'end of the dystrophin pre-mRNA exon 51 and the nucleotides at the 5' end of the dystrophin pre-mRNA exon 51.
[0121] [00121] Antisense Tricycle-Phosphorothioate DNA Oligonucleotides for the Treatment of Spinal Muscular Atrophy
[0122] [00122] Within other modalities, the present disclosure provides AON of tc-DNA-PS that can be adequately employed for the treatment of Spinal Muscular Atrophy (SMA). SMA is caused by mutations in both copies of the SMN1 gene, which in a normal cell is characterized by the presence of exons 7 and 8 in the fully processed mRNA. A second gene present in humans in varying copy numbers, SMN2, carries a silent mutation in exon 7 that alters an exonic splice enhancing sequence. As a consequence, SMN2 splicing is altered compared to SMN1 and only 10% of a normal full length SMN protein is transcribed from that gene while other non-functional SMN2 transcripts are deleted for exon 7. Low abundance of the normal full length SMN2 transcript cannot fully compensate for the lack of the SMN 1 transcript, thus causing the disease. By masking an intronic silencing sequence (ISS) and / or a terminal rod-loop (TSL) within an SMN2 pre-mRNA, it is expected that the tc-DNA-PS AON described in this document can facilitate the inclusion of SMN2 exon 7 in a processed SMN2 pre-mRNA, which is translated into a fully functional SMN2 protein that is identical to the SMN1 protein and therefore can compensate for the loss of the functional SMN1 protein. When expressed in vivo, the increased amounts of SMN2 protein can at least partially reverse Spinal Muscular Atrophy which is caused by mutations in the SMN1 gene.
[0123] [00123] Thus, the present disclosure provides tc-DNA-PS AON to facilitate the inclusion of exon 7 during the processing of an SMN2 pre-mRNA in which the tc-DNA-PS AON has 6 to 22 tricycle nucleotides in length, particularly between 8 to 20 tricycle nucleotides, more particularly between 10 to 18 tricycle nucleotides in length and where the tc-DNA-PS AON is complementary to an SMN2 pre-mRNA intronic silencing sequence (ISS) or a terminal rod-loop (TSL). Such a tc-DNA-PS AON can be between 13 and 17 nucleotides, between 12 and 16 nucleotides or between 13 and 15 nucleotides.
[0124] [00124] Exemplified herein are the tc-DNA AONs which comprise a sequence of 15 nucleotides 5'-CTTTCATAATGCTGG-3 '(SMN2i7 (10; 25), SEQ ID NO: 7), such tc-DNA AONs are complementary to an SMN2 ISS pre-mRNA and that can be used to facilitate the inclusion of exon 7 in a processed SMN2 mRNA. Also exemplified in this document are the tc-DNA-PS AONs that comprise the 13 nucleotide sequence 5'-CTTTCATAATGCTGG-3 '(SMN2e7 (39; 51), SEQ ID NO: 8), such tc-DNA- PS are complementary to a SMN2 TSL2 pre-mRNA and can also be used to facilitate the inclusion of exon 7 in a processed SMN2 mRNA.
[0125] [00125] Antisense Tricycle-Phosphorothioate DNA Oligonucleotides for the Treatment of Steinert's Myotonic Dystrophy
[0126] [00126] Within still further modalities, the present disclosure provides AON of tc-DNA-PS that can be properly employed for the treatment of Steinert's Myotonic Dystrophy that results from CUG amplifications at the 3 'end of the mRNA encoding DM1. Mutated DM1 mPvNAs that contain excessive CUG amplifications are believed to be sequestered in the nucleus and accumulate to form nuclear foci. These foci are stable and are believed to be linked to factors involved in the splicing mechanisms thus largely affecting the transcriptome. As part of the present disclosure, it is hoped that tc-DNA-PS AONs can be employed to target the CUG sequences and facilitate the destruction of the mutated DM1 mRNA and / or prevent the sequestration of nuclear proteins to the expanded CUG repeats thus leading to the release of splicing factors and the removal of nuclear foci. Without adhering to a particular mechanistic theory, it is further believed that the tc-DNA-PS AONs disclosed herein can facilitate the destruction of mRNAs that contain excessive CUG amplifications.
[0127] [00127] Thus, tc-DNA-PS AON are described that can be properly employed to facilitate the destruction of a mutated DM1 mRNA that comprises excessive CUG amplifications. Such tc-DNA-PS AONs comprise 9 to 27 tricycle nucleotides, wherein the tc-DNA AON is complementary to a mutated DM1 mRNA comprising one or more 3 'CUG amplifications and in which the tc-DNA AON -PS can facilitate the destruction of DM1 mRNA. Depending on the precise application contemplated, AON of tc-DNA-PS can comprise between 3 and 9; between 4 and 8; or 5, 6, or 7 contiguous repeats of the 5'-CAG-3 'nucleotide sequence (SEQ ID NO: 9). An exemplary tc-DNA-PS AON expected to facilitate the destruction of a mutated DM1 comprises the sequence of 15 nucleotides 5'-CAGCAGCAGCAGCAG-3 '(DM1 (CAG5), SEQ ID NO: 10). Another exemplary tc-DNA-PS AON expected to facilitate the destruction of a mutated DM1 comprises the sequence of 15 nucleotides: 5'-CAGCAGCAGCAGCAGCAGCAG -3 '(DM1 (CAG7), SEQ ID NO: 11).
[0128] [00128] Tricycle-phosphorothioate Antisense Oligonucleotides for the Treatment of Heart Diseases
[0129] [00129] The most common genetic cause of hypertrophic cardiomyopathy (HCM) are mutations in cardiac myosin-binding protein C (for review see: Schlossarek, S, et al. J Mol Cell Cardiol 50 (2011) 613 to 620). Very recently, the exon hop was applied in vitro to modify a mutated cMyBP-C molecule in mouse myocytes cMyBP-C ki (Gedicke, C, Behrens-Gawlik, V, Dreyfus, PA, Eschenhagen, T, Carrier, L. Specific skipping of exons using antisense oligoribonucleotides results in novel molecule in cMyBP-C knock-in mouse myocytes. Circ 201; 122 (Suppl): A 19079). Due to its absorption into cardiac tissue after systemic delivery, tc-DNA-PS could be adequately employed to correct the mutated cMyBP in cardiac tissue. Certainly, the present tc-DNA-PS are also anticipated to be useful for the correction of other proteins in cardiac tissue.
[0130] [00130] Tricycle-phosphorothioate Antisense Oligonucleotides for the Treatment of CNS diseases
[0131] [00131] Unexpectedly, it has been shown that the tc-DNA-PS molecules of the invention cross the blood-brain barrier. Accordingly, the present invention relates to a method for providing the targeting of an oligonucleotide to the CNS, comprising administering to a subject in need of it a tc-DNA-PS oligonucleotide. In addition, the invention also relates to a method for the treatment of a disease that affects the CNS of an individual in need thereof, which comprises administering to said individual, particularly a human individual, a tc-DNA-PS molecule of the invention. The tc-DNA-PS is complementary to a defined target sequence so that the interaction between the administered tc-DNA-PS and the target sequence provides an effective treatment of said disease. In a particular embodiment, the nucleic acid molecules of the invention can be used to treat diseases that affect both the muscles and the CNS. As mentioned, although Duchenne's muscular dystrophy is mainly characterized by the observed muscle dysfunction, about a third of DMD patients also exhibit cognitive impairment suggesting a notable neuronal and brain function disorder. The nucleic acid molecules of the invention can therefore be used to restore neuronal and brain function resulting from the abnormal dystrophin.
[0132] [00132] Additionally, nucleic acid molecules of the invention can be used to treat diseases for which CNS disorders are the main or one of the main resources. For example, the principles described above for restoring a functional protein (either by exon skipping or exon inclusion) or to destroy a particular pre-mRNA can be transposed to treat diseases such as spinal muscle amyotrophy, myotonic dystrophy or Huntington.
[0133] [00133] EXAMPLES
[0134] [00134] The above disclosure generally describes the present disclosure, which is further exemplified by the following examples. These specific examples are described for illustrative purposes only and are not intended to limit the scope of this disclosure. Although specific targets, terms and values have been used in this document, such targets, terms and values will also be understood to be exemplary and not to limit the scope of this disclosure.
[0135] [00135] Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects one in 3500 male live births (Emery. Neuromuscul. Disord. 1991). It is caused by mutations in the gene that encodes dystrophin, a large protein (427 kDa) found in a variety of tissues, especially in striated muscle fibers and neurons in particular regions of the central nervous system (Kunkel et al, PNAS. 1985; Muntoni F et al, Lancet Neurol. 2003). Dystrophin is located close to the inner surface of the plasma membrane, connecting the actin cytoskeleton to the extracellular matrix through a glycoprotein complex associated with membrane dystrophin (Culligan et al, 1988). The lack of dystrophin makes muscle fibers particularly vulnerable to mechanical stress and undergo recurrent necrosis cycles. As a result, patients exhibit progressive weakness of skeletal muscles that are, over time, replaced by adipofibrotic tissue, leading to loss of locomotion around the age of twelve and therefore premature death is caused either by respiratory failure or cardiomyopathy between the second and fourth decade. In addition, about a third of DMD patients also exhibit cognitive impairment suggesting a notable disorder of neuronal and brain function (Bresolin et al, Neuromuscul. Disord. 1994).
[0136] [00136] Full-length dystrophin, translated from a main 14 kb mRNA transcript made of 79 exons, is a modular protein that can successfully withstand the deletion of multiple exons as long as the open reading phase is preserved ( Koenig et al, Cell, 1987). This phenomenon occurs in clinically milder Becker muscular dystrophy (BMD) disease, in which the deletions that keep the reading phase open lead to the synthesis of truncated semifunctional forms of dystrophin (Monaco et al. Genomics. 1988). Therefore, it was proposed, fifteen years ago, that interfering with the process of splicing of the elected exons with the use of antisense oligonucleotides (AON) may be an adequate therapeutic approach for DMD (Matsuo M. Brain Dev. 1996).
[0137] [00137] Two types of compounds have been tested extensively for antisense-induced exon hopping, 2'-0-methyl modified ribose oligomers with a full-length phosphorothioate backbone (20Me-PS) and morpholino oligomers phosphorodiamidate (PMO). Both types of antisense molecules have been shown to rescue dystrophin in skeletal muscle after systemic delivery in animal models of DMD and more recently in clinical trials. In the current situation, clinical tests using systemic administration of 2'OMe-PS and exon 51 of PMO targeting of dystrophin pre-mRA have been well tolerated without any serious drug-related adverse events (van Deutekom et al., New. Engl. J; Med. 2007; Kinali et al., Lancet Neurol. 2009; Goemans et al, New. Engl. J. Med. 2011; Cirak et al., Lancet 2011). However, these compounds are a major limitation, which is that they do not effectively target the heart muscle and do not cross the blood-brain barrier.
[0138] [00138] Here, we show that the systemic delivery of antisense oligomers made of nucleotide analogs of tricyclo-DNA (tc-DNA) allows rescue of dystrophin in skeletal muscles in the mdx mouse model. In addition, the substitution of sulfur for oxygen in the phosphate ester main chain gave new properties to tc-DNA antisenses that were crucial for its biodistribution after systemic administration. In fact, tc-DNA oligomers that contain phosphorothioate (PS) could now efficiently target the heart muscle and, furthermore, cross the blood brain barrier to rescue dystrophin that has mutated in the heart and central nervous system.
[0139] [00139] Material and Methods:
[0140] [00140] Tricycle-DNAs.
[0141] [00141] The synthesis of tc-DNA with phosphorothioate followed classical procedures in the synthesis of solid phase oligonucleotides according to the phosphoramidite approach. The synthesis cycle in which an additional unit is attached to the growing chain consists of four sequential steps (a-d). After the chain assembly, the oligonucleotide is detached from the solid and unprotected support in the usual way (NH3 cone, 55 ° C, 16 hours). Glass with long-chain aquilamine-controlled pore (LCAA-CPG), to which the first t-nucleoside is attached by means of a succinyl ligand, is used as a solid support. The syntheses were generally carried out on a scale of 1, 3 or 10 μmoles in a DNA synthesizer plus Pharmacia gene assembler. The Tc-PS-oligonucleotides were synthesized with a 5 'terminal phosphate or thiophosphate group to ensure chemical stability of the 5' end. The conditions for each step a) to d) are given below and are optimized for a synthesis of 10 μmoles.
[0142] [00142] Detritilation: Leveled with 3% dichloroacetic acid in 1,2-dichloroethane (DCE) for 1.5 minutes. Then washed with DCE and CH3CN.
[0143] [00143] Coupling: Phosphoramidite solution (0.1 mM in CH3CN, 400 ml) and activator 5-ethylthietrazole (ETT, 0. 25M in CH3CN, 600 ml) is applied to the solid support. Docking time: 9 minutes. Then it is washed with CH3CN.
[0144] [00144] Sulfurization: Bis (phenylacetyl) disulfide (PADS) in dry pyridine / CH3CN 1/1 (0.2 M) is leveled on the solid support for 3 minutes. Then washed with CH3CN.
[0145] [00145] Capping: Unreacted 5'-hydroxyl groups are capped using Cap A (4-dimethylaminopyridine (DMAP, 0, 5 M) in CH3CN) and Cap B solution (acetic anhydride (AC20), collidine in CH3CN (2: 3: 5)) for 20 seconds each. Then washed with CH3CN.
[0146] [00146] The antisense sequence to rescue the mdx dystrophin pre-mRNA was 15 monomers in length and targeted the exon 23 donor splice site (M23D (+ 2-13)).
[0147] [00147] 5’-AACCTCGGCTTACCT-3 ’(SEQ ID NO: 1)
[0148] [00148] The other antisense sequences in this document have also been synthesized according to this method.
[0149] [00149] Animal experiments
[0150] [00150] Adult mdx mice (6 to 8 weeks of age) were injected intramuscularly, intravenously or subcutaneously with tc-DNA or tc-DNA-PS as indicated in the results section under general anesthesia with isofluorane use.
[0151] [00151] DKO mice are generated by crossing (utr +/-, dys - / -) mice, which were obtained by crossing utr - / - mice with mdx mice (Deconinck, AE, Rafael, JA, Skinner, JA, Brown, SC, Potter, AC, Metzinger, L., Watt, DJ, Dickson, JG, Tinsley, JM and Davies, KE (1997) Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell, 90 , 717 to 727.) TcDNA were delivered weekly to dKO mice at a dose of 200 mg / kg / week by intravenous (IV) injections in the tail vein and subcutaneous (Sc) injections alternatively with mice under general anesthesia. The treated mice were killed at various time points as indicated in the results section for CO2 inhalation. The muscles were quickly frozen in isopentane cooled by liquid nitrogen and stored at -80 ° C before further analysis. All dKO experiments were performed at the Biomedical Science Building, University of Oxford, Oxford, United Kingdom, and performed according to the guidelines and protocols approved by the Home Office.
[0152] [00152] Analysis of muscle function
[0153] [00153] Functional grip strength analysis was performed on treated and control mice at 12 weeks of age using a commercial grip strength monitor (Chatillon, UK). Each mouse was kept 2 cm from the base of the tail, allowed to grab a bar attached to the device with its front legs and gently pulled until they released the bar. The strength exerted was recorded from 4 sequential tests, with an average of 1 minute interval. Specific strength and strength drop were measured from the long finger extensor muscle (EDL) dissected from the rear leg of the treated and control mice. During dissection and experiments, the muscles were washed in an oxygenated Krebs-Hensley solution (95% O2-5% CO2) composed of (mM): NaCl, 118; NaHCO3, at 24, 8, KC1, at 4, 75; KH2PO4, at 1.18; MgSO4, at 1.18; CaCl2, at 2.54; glucose, at 10. Contractile properties were measured as previously described (Goyenvalle, A., Babbs, A., Powell, D., Kole, R., Fletcher, S., Wilton, SD and Davies, KE (2010) Prevention of dystrophic pathology in severely affected dystrophin / utrophin-deficient mice by morpholino-oligomer-mediated exon-skipping. Mol. Ther., 18, 198 to 205.)
[0154] [00154] Monitoring of activity in the open field
[0155] [00155] The Linton AM 1053 X, Y, Z IR activity monitors were used to monitor open field activity in dKO mice. The mice were acclimatized in empty cages for 90 minutes the day before the actual data collection. Data were collected every 10 minutes over a period of 90 minutes for 3 consecutive days. The first 3 of the 9 records on each day were discarded by analysis. 22 different activity parameters were measured for each mouse, with total distance covered, total activity, vertical exploration time and total movement counts considered to be the best parameters for monitoring behavioral activity.
[0156] [00156] Immunohistochemistry and histology
[0157] [00157] 8 μm sections were cut from at least two thirds of the length of the anterior tibial muscle, gastrocnemius, quadriceps, gluteus, biceps, triceps, diaphragm and cardiac at 100 μm intervals. The sections of intervention muscles were collected for subsequent RT-PCR analysis. Routine hematoxylin and eosin staining was used to examine general muscle morphology. The cryosections were then examined for expression of dystrophin using the rabbit polyclonal antibody DYS (Novocastra, United Kingdom), which was then detected by Alexa 488 of goat anti-rabbit IgGs.
[0158] [00158] RNA isolation and RT-PCR analysis
[0159] [00159] Total RNA was isolated from the intervention muscle sections collected during cryosection using TRIzole reagent according to the manufacturer's instructions (Invitrogen, UK). 200 ng aliquots of total RNA were used for RT-PCR analysis using the Access RT-PCR System (Promega) in a 50 μ reaction! using Ex 20Fo (5'-CAGAATTCTGCCAATTGCTGAG-3 '; SEQ ID NO: 12) and Ex 26Ro (5'-TTCTTCAGCTTGTGTCATCC-3'; SEQ ID NO: 13) primers. The cDNA synthesis was performed at 45 ° C for 45 minutes, directly followed by the primary PCR of 30 cycles of 94 ° C (30 seconds), 58 ° C (1 minute) and 72 ° C (2 minutes). The microliters of these reactions were then reamplified in PCRs nested for 22 cycles of 94 ° C (30 seconds), 58 ° C (1 minute) and 72 ° C (2 minutes) using the Ex 20Fi (5'- CCCAGTCTACCACCCTATCAGAGC-3 '; SEQ ID NO: 14) and Ex 26Ri (5'-CCTGCCTTTAAGGCTTCCTT-3'; SEQ ID NO: 15). PCR products were analyzed on 2% agarose gels.
[0160] [00160] Detection of dystrophin mRNA with exon 23 skipped in mdx central nervous system
[0161] [00161] The mdx mice were treated biweekly with subcutaneous and intravenous injections of M23D (+ 2-13) (main chains of tc-DNA or tc-DNA-PS) for 8 weeks, with a dose of 100 mg / kg of weight body. One week after the last injection, the brains were dissected and processed by detecting dystrophin mRNA with skipped exon 23. RNA samples were analyzed by nested RT-PCR using primers (Ex 20Fo (external) / Ex 20Fi (internal) that annex exon 20 and Ex 26Ro / Ri22-24 that annex exon 26 and junction 2224, respectively) allowing specific recognition of the skipped messenger as a 398 bp fragment (Ri22-24 5'-TTATGTGATTCTGTAAATTC-3 'SEQ ID NO: 16). Note that since the Ri primer specifically annexes the exon 22-exon 24 limit, the untapped dystrophin mRNA is not amplified and the 398-bp band can be detected only in samples containing dystrophin mRNAs without exon 23.
[0162] [00162] Quantification of exon 23 hop by quantitative PCR
[0163] [00163] RNA was isolated from mouse tissue as described above. Contaminating DNA was removed from RNA preparations using the Turbo DNA-free system (Ambion). Aliquots of 1 μg of RNA treated with DNase were then subjected to reverse transcription using the First Strand synthesis system (Invitrogen) with random hexamers according to the manufacturer's instructions. Quantitative PCR was performed using Taqman assays that were designed against exon 4-5 or exon 22-24 matrices using the Custom Assay Design Tool (Applied Biosystems) as described in Goyenvalle et al, Rescue of severely affected dystrophin / utrophin deficient mice through scAAV-U7snRNA-mediated exon skipping; Human Molecular Genetics, 2012, Volume 21, no 11 25592571. An inventoried 18S assay was used as an endogenous control (Applied Biosystems, 4310893E). 50 ng cDNA was used as a reaction input and all assays were run in singleplex. The tests were performed under fast cycle conditions in an Applied Biosystems StepOne Plus thermocycler, and all data were analyzed using the comparative Ct method using the associated StepOne analytical software. For a given sample, delta-Ct values from exon 4-5 and exon 22-24 assays were used to calculate a relative abundance of total dystrophin and skipped exon 23 mRNA, respectively. The exon jump 23 was then expressed as a percentage against total dystrophin, as indicated by the exon 4-5 expression level.
[0164] [00164] Western blot analysis
[0165] [00165] Total protein was extracted from muscle samples with buffer containing 250 sucrose, 10 mM Tris-HCl, pH 6, 7, 20% dodecyl sodium sulfate, 20% glycerol, 10% β- mercaptoethanol, 12.5% continuous buffer (Life Technologies) and a mixture of protease inhibitors (Roche). The samples were denatured at 95 ° C for 5 minutes and centrifuged. Then, the aliquot was precipitated using the Compat-Able Protein Assay Preparation Reagent Set and quantified with the BCA Protein Assay kit (Pierce) and 50 μg or 100 μg of protein was loaded onto a polyacrylamide gel (NuPage 4 to 12% Bis -Tris, Life Technologies). The gels were electrophoresed for 4 to 5 hours at 130V and transferred to a nitrocellulose membrane overnight at 100 mM. The blots were blocked for 1 hour with 10% skimmed milk in PBS-Tween buffer (PBST). Dystrophin and α-Actinin proteins were detected by probing the membrane with a 1: 50 solution of primary antibody NCL-DYS1 (monoclonal antibody for dystrophin R8 repeat; NovoCastra) and a 1: 5,000 dilution of primary α-actinin antibody ( Santa Cruz Biotechnology), respectively, followed by incubation with a secondary antibody conjugated to mouse horseradish peroxidase (1: 15. 000). Western blots were developed with improved chemiluminescence (Thermo Scientific) and ECL Analysis System (ECL-Plus; GE Healthcare). The actin bands were used to verify that the protein load was correct. The membranes were converted into numerical images by scanning and the band intensities were analyzed using the ImageJ 1.46r software (http: // rsb. Info. Nih. Gov / ij /).
[0166] [00166] Quantification of serum biomarker levels
[0167] [00167] Blood samples were collected from tail bleeds under general anesthesia. The analysis of levels Creatine kinase (CK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was performed by the pathology laboratory (Mary Lyon Center, Medical Research Council, Harwell, Oxfordshire, United Kingdom).
[0168] [00168] Statistical Analysis
[0169] [00169] All results are expressed as mean values ± SEM unless otherwise stated. The differences between treated and control cuts were determined using an unpaired student t test.
[0170] [00170] Example 1 - in vivo evaluation of tc-DNA-PS antisense oligonucleotides for the treatment of dystrophin-mediated muscular dystrophy
[0171] [00171] Adult mdx mice were treated systemically for 12 weeks using subcutaneous and / or intravenous injections of oligomer tc-DNA-PS M23D (+ 2-13) at either 200 or 50 mg / kg body weight per week . Two weeks after the last injection, the muscles were harvested and the RNA samples were analyzed by RT-PCR nested with primers in exons 20 and 26 of the dystrophin gene. Figure 3A shows the detection of dystrophin mRNA with exon jump 23 in a number of skeletal muscles of treated animals. The 903-bp band corresponds to the untapped dystrophin mRNA involving the non-sense mdx mutation, while the smaller 688-bp fragment corresponds to the exon 23 mRNA. It is notable that systemic treatment with the phosphorothioate-containing oligomer induces significant rescue dystrophin mRNA in various skeletal muscles (ie, anterior tibial muscle, gastrocnemius, quadriceps, gluteus, triceps, biceps, diaphragm), including respiratory muscles, as well as in heart muscle. Consistent with the generation of jump transcripts, the dystrophin protein was readily detected both by Western blot analysis (Figure 3B) and by immunofluorescence in tissue sections (Figure 4). The dystrophin levels mirrored those of the rescued mRNA and the jump procedure generated a species with immunoreactive protein with a mobility around 427 kDa. The expected 8 kD difference between wild type and rescued proteins could not be resolved in the type of gel used in this study. Importantly, both delivery modes, intravenous and subcutaneous, gave rise to rescue of similar diffused dystrophin in mdx as shown in Figure 5. Whatever the systemic delivery mode, oligonucleotides made using the tc-DNA main chain normal (ie, with normal phosphodiester internucleoside binding) were unable to significantly target the heart muscle. This was only achieved with the phosphorothioate (-PS) backbone as illustrated in Figure 6. The phosphorothioate modification that confers substantial pharmacokinetic benefit and it was investigated whether such an adaptation could allow oligonucleotides to cross the blood-brain barrier. In fact, it has been shown that oligonucleotides using the normal tc-DNA main chain rescue mRA dystrophin when delivered to the cerebrospinal fluid after stereotactic injection into the cisternal magna, which suggests that they could cross the ependymal epithelium. However, such compounds were inefficient when delivered intravenously and / or subcutaneously, demonstrating that they could not cross the blood-brain barrier (Figure 7). In fact, this was only successfully achieved when using the phosphorothioate (-PS) forms of tc-DNAs (Figure 8), thus demonstrating their ability to access all major tissues in which dystrophin had to be re-stored ideally: skeletal muscles, heart and CNS.
[0172] [00172] The therapeutic effect of systemic delivery of tc-DNA-PS (M23D + 2-13) was confirmed by the significant decrease in creatine kinase levels in the serum of treated animals, indicating that the amount of dystrophin rescued was appropriate to protect the fibers from exercise-induced damage without obvious toxicity as estimated by the blood levels of ALT and AST, which did not increase whatever the concentration of oligomers (Figure 9A and B). Muscle enhancement was also assessed by testing the specific strength of treated muscles, which was significantly improved (Figure 9C). More importantly, the percentage of strength loss, a characteristic feature of dystrophic muscle assessed by measuring strength deficit following a series of eccentric contractions, was reduced in treated animals, confirming that muscle fibers in treated animals were much more resistant ( Figure 9D).
[0173] [00173] Clinical relevance has been estimated in the transgenic mouse dKO, a more severe DMD model, which lacks both dystrophin and utrophin, resulting in progressive muscle weakness, impaired mobility and premature death. As for mdx, systemic treatment of dKO with tc-DNA-PS allowed significant dystrophin rescue in all tissue compartments (Figure 10). The percentage of exon jump was assessed using quantitative RT-PCR at different time points after the start of treatment showing that there was a cumulative effect on the duration of treatment. The mRNA rescue was almost complete in the diaphragm after 20 weeks and a person could expect that the other skeletal muscles would reach that jump level within 40 weeks and later to the heart and brain for which the oligomer lift seems to be lower (Figure 11) . However, rescue levels of dystrophin in dKO after 12 weeks of treatment provide significant clinical benefit. The treated mice did not show the characteristic kyphosis, CK levels were decreased while the mice were more physically active and exhibited improved physiological parameters (Figure 12). Although pharmacokinetic studies have shown that the oligonucleotides disappeared from the serum within minutes after the intravenous injection (Figure 13A), it appears that they have a prolonged effect once inside the target tissue. This is suggested by the fact that the jump levels were found to be about half of their maximum value 13 weeks after the end of treatment (Figure 13B). This prolonged effect is confirmed in the results shown in Figure 14. It is likely that tc-DNA-PS are stable in cells and that they can be re-employed over time, thereby limiting the need to fill tissues as would often be necessary if these oligonucleotides were destroyed or titrated by their mRNA targets.
[0174] [00174] Example 2: Effect of delivering a tc-DNA-PS (ISS7) that targets an SMN2 exon 7
[0175] [00175] The SMA mouse model (FVB. Cg-Tg (SMN2) 2Hung Smn1tm1Hung / J) was used. Type III SMA mice (FVB. Cg-Tg (SMN2) 2Hung Smn1tm1Hung / J) are defeated to Smn (Smn1 - / -) and contain an SMN2 transgene made of two tandem copies of the human SMN2 gene. These animals exhibit typical features that include necrosis of the cause starting at around one month of age. Such necrosis progressively extends to the ears and feet and later in life that these animals present with muscle weakness. The photograph in Figure 15 shows 3 type III individuals (one month old). The individual at the top is the untreated control; the other two were treated with tc-DNA-PS (ISS7): they received a single ICV (intracerebroventricular) injection at birth (5 μl containing 20 μg of tc-DNA-PS (ISS7)) and repeated SC (subcutaneous) injections once a week at a dose of 200 mg / kg.
[0176] [00176] It is concluded that tc-DNA-PS oligomers represent a possible drug candidate for SMA therapy. Furthermore, as this type of oligonucleotide can spontaneously cross the blood-brain barrier (see section mdx), it is likely that tc-DNA-PS would not necessarily require intracerebral administration to effectively redirect SMN2 splicing in CNS.
[0177] [00177] Example 3: Evaluation of tc-DNA and tc-DNA-PS for DM1
[0178] [00178] DM1 myoblasts with 800 CTG replications were transfected with increasing concentration of tc-DNA-CAG7 (SEQ ID NO: 11). After 3 days in culture, the expression of both normal and mutant CUGexp-DMPK (myotonic dystrophy-protein kinase) mRNAs was analyzed by Northern blot. The mutant vs. CUGexp-DMPK ratio Normal DMPK mRNAs were quantified. The dose-dependent decrease of the mutant CUGexp-DMPK mRNA without suspending the normal DMPK mRNA showed that treatment with the oligonucleotide results in the specific destruction of the mutant CUGexp-DMPK (see Figure 16)
[0179] [00179] In another experiment, DM1 myoblasts with expanded CTG (> 800 CTG) were transfected with 10 μg of tc-DNA-PS-CAG7. After 3 days in culture, expression of normal and mutant CUGexp-DMPK mRNAs was analyzed by Northern blot. The mutant vs. CUGexp-DMPK ratio Normal DMPK mRNAs were quantified (Figure 17 top). Nuclear clusters of expanded CUG RNAs (foci) were detected by FISH and the number of cells without CUGexp-RNA nuclear clusters was quantified (Figure 17 bottom). The results showed that DM1 myoblasts transfected with the oligonucleotide have i) reduced level of mutant CUGexp-DMPK mRNAs without any change in normal DMPK mRNAs; ii) increased number of cells without nuclear aggregates.
[0180] [00180] The effect of the tc-DNA-PS-CAG7 oligonucleotide was then evaluated in vivo. Since the oligonucleotide targets the CUG-expanded RNA of DMPK transcripts without affecting normal DMPK transcripts in DM1 muscle cells, it was decided to estimate the effect of the oligonucleotide on a DM1 model mouse expressing CUG-expanded RNA in the region without 3 'coding of the human skeletal actin (HSA) gene. This mouse model DM1 has already been used to estimate both CAG8 morpholino and CAG8 2'-O-Me ASO because it showed poor regulation of alternative splicing of several RNA transcripts as well as myotonia, which results from pre- ClC-1 mRNAs.
[0181] [00181] The tibial anterior TA muscles of HSA-LR mice expressing 250CTG in the 3 'UTR of the human skeletal actin (HSA) gene were injected with increasing concentration of tc-DNA-PS-CAG7. The contralateral TA muscles were injected with saline and used as a control. After 2 weeks, the expression of HSA and MSA mRNAs (mouse skeletal actin) was analyzed by Northern blot. The rate of HSA vs. mRNAs MSA was quantified. Figure 18 shows that an intramuscular injection of the oligonucleotide results in a marked decrease in RNAs-CUGexp.
[0182] [00182] TA muscles of HSA-LR mice that express 250CTG in the 3'UTR of the human skeletal actin gene (HSA) were also injected with 30 μg of tc-DNA-PS-CAG7. The contralateral TA muscles were injected with saline and used as a control. After 1 and 2 weeks, the expression of HSA and MSA mRNAs (mouse skeleton actin) was analyzed by Northern blot. The rate of HSA vs. mRNAs MSA was quantified. Figure 19 shows that a decreased level of RNAs-CUGexp following intramuscular injection of the oligonucleotide has already been observed after one week.
[0183] [00183] Finally, the GA gastrocnemius muscles of HSA-LR mice expressing 250CTG in the 3 'UTR of the human skeletal actin gene (HSA) were injected with 90 μg of tc-DNA-PS-CAG7. The contralateral GA muscles were injected with saline and used as a control. The expression of HSA mRNAs and MSA (mouse skeletal actin) mRNAs was analyzed by Northern blot after 2, 4 and 8 weeks. The rate of HSA vs. mRNAs MSA was quantified. Figure 20 shows that an intramuscular administration of the oligonucleotide causes an efficient destruction of RNAs-CUGexp and the effect is sustained between 4 to 8 months after treatment.

权利要求:
Claims (14)
[0001]
Nucleic acid molecule characterized by the fact that it comprises tricyclonucleosides joined by internucleoside phosphorothioate bonds (3'-OPS-0-5 'bonds).
[0002]
Nucleic acid molecule according to claim 1, characterized in that it comprises between 3 and 50 nucleotides.
[0003]
Nucleic acid molecule according to claim 1 or 2, characterized in that it is complementary to a target sequence.
[0004]
Nucleic acid molecule according to claim 3, characterized in that the nucleic acid molecule is an antisense oligonucleotide complementary to a portion and an RNA encoded by a gene.
[0005]
Nucleic acid molecule according to claim 1, characterized in that it comprises or consists of the sequence selected from the group consisting of SEQ ID NOs: 1 to 11.
[0006]
Method for the synthesis of a tricyclo-phosphorothioate DNA molecule, the method being characterized by the fact that it comprises: a) providing a first tricyclo-nucleoside attached to a solid phase support, wherein said first nucleotide has a protected 5'-OH group; b) deprotecting the 5 'group to form a free 5'-OH group; c) reacting the free 5'-OH group with a 5'-protected 3'-O-cyanoethyl-N, N-diisopropylaminophosphoramidite tricyclonucleoside monomer to form an internucleoside phosphoramidite bond between the first and a second tricyclonucleosides; and d) sulfurize the internucleoside phosphoramidite group to form a phosphorothioate internucleoside bond between the first and the second tricyclonucleosides.
[0007]
Pharmaceutical composition characterized by the fact that it comprises a nucleic acid molecule, as defined in any one of claims 1 to 4, in a pharmaceutically acceptable carrier.
[0008]
Pharmaceutical composition according to claim 7, said composition being characterized by the fact that it is an injectable composition, in particular, a composition for intravenous injection.
[0009]
Nucleic acid molecule according to any one of claims 1 to 5, characterized by the fact that it is for use as a medicament.
[0010]
Nucleic acid molecule according to any one of claims 1 to 4, characterized by the fact that it is for use in the treatment of heart disease.
[0011]
Nucleic acid molecule according to any one of claims 1 to 5, characterized by the fact that it is for use in the treatment of a neuromuscular or musculoskeletal disease.
[0012]
Nucleic acid molecule according to any one of claims 1 to 4, characterized by the fact that it is for use in the treatment of a disease of the central nervous system.
[0013]
Nucleic acid molecule according to any one of claims 10 to 12, characterized by the fact that heart, CNS, neuromuscular or musculoskeletal disease results from an alteration of a gene, wherein said alteration is a mutation without changing the exon reading phase, a mutation that disrupts the translational reading phase of the gene, and tc-DNA facilitates the hopping of an exon in order to restore the reading phase; a deleterious mutation that can be compensated for by the inclusion of an atypical exon in the mRNA encoded by said gene and the tc-DNA is complementary to an ISS or TSL present in a pre-mRNA encoded by said gene and facilitates the inclusion of an atypical exon, or a mutation that results in the presence of deleterious 3 'CUG amplification (s) in an mRNA encoded by said gene.
[0014]
Nucleic acid molecule according to claim 11, characterized by the fact that it is for the treatment of Duchenne Muscular Dystrophy, Spinal Muscular Atrophy or Steinert's Myotonic Dystrophy.

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Leumann et al.0|des brevets | GG 0 000 000 L0 | LLLLLGGG GGGGGGGG LLLLL GGGGGGGG

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EP2581448B1|2015-01-28|

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BR112014009066A2|2017-04-18|

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BR112014009066A8|2018-01-09|

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法律状态:
2018-05-08| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-09-17| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-02-27| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-03-10| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: C12N 15/113 , C12N 15/11 , A61K 31/712 Ipc: C12N 15/113 (2010.01), A61K 31/712 (2006.01), A61K |

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2020-06-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-08-25| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 12/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-02-23| B16C| Correction of notification of the grant|Free format text: REF. RPI 2590 DE 25/08/2020 QUANTO AO TITULAR. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161546942P| true| 2011-10-13|2011-10-13|

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EP11185129.1|2011-10-13|

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EP11185129.1A|EP2581448B1|2011-10-13|2011-10-13|Tricyclo-phosphorothioate DNA|

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PCT/EP2012/070349|WO2013053928A1|2011-10-13|2012-10-12|Tricyclo-phosphorothioate dna|

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