Compounds and methods for modulating smn2
a technology of smn2 and rna, applied in the field of compounds, methods and pharmaceutical compositions for modulating smn2 rna, can solve the problems of inefficient inclusion of exon 7 in smn2 transcripts, and achieve the effects of reducing neuromuscular activity, reducing muscle strength, and reducing the inability or reducing the ability to stand upright, walk, and/or stand
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example 1
Modified Oligonucleotides Complementary to a Human SMN2 Nucleic Acid
[0286]Modified oligonucleotides complementary to a human SMN2 nucleic acid were designed and synthesized as indicated in the tables below.
[0287]The modified oligonucleotides in the tables below are 16, 17, 18, 19, or 20 nucleosides in length, as specified. The modified oligonucleotides comprise 2′-MOE sugar moieties, 2′-NMA sugar moieties, cEt sugar moieties, 2′-OMe sugar moieties, and / or 2′β-D-deoxyribosyl sugar moieties, as specified. Each internucleoside linkage throughout the modified oligonucleotides is either a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage, as specified. Cytosines are either non-methylated cytosines or 5-methyl cytosines, as specified.
[0288]Each modified oligonucleotide listed in the tables below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NT_006713.14 truncated from nucleotides 19939708 to Ser. No. 19 / 967,777), unless specifically stated...
example 2
of Modified Oligonucleotides Complementary to Human SMN2 in Transgenic Mice, Single Dose (35 μg)
[0307]Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice. Taiwan strain of SMA Type III mice were obtained from The Jackson Laboratory (Bar Harbor, Me.). These mice lack mouse SMN and are homozygous for human SMN2 (mSMN− / −; hSMN2+ / +; FVB.Cg-Tg(SMN2)2HungSMN1tm1Hung / J, stock number 005058; Bar Harbor, Me.), or are heterozygous for mouse SMN and heterozygous for human SMN2(mSMN+ / −; hSMN2+ / −; FVB.Cg-Tg(SMN2)2HungSMN1tm1Hung / J) obtained by breeding HOM / HOM (stock #00005058) to FVB / NJ (Stock #001800).
Treatment
[0308]Homozygous or heterozygous transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of 35 μg of modified oligonucleotide. Comparator Compound Nos. 387954, 396442, and 396443 were also tested in this assay. A group of 4 mice received PBS as a negative control.
RNA Analysis
[0309]Two weeks post t...
example 3
of Modified Oligonucleotides Complementary to Human SMN2 in Transgenic Mice, Single Dose (15 μg)
[0311]Activity of selected modified oligonucleotides described above was tested in human SMN2 transgenic mice essentially as described above in Example 2. Comparator Compound Nos. 396443 and 819735 were also tested in this assay. The transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of 15 μg of modified oligonucleotide. A group of 4 mice received PBS as a negative control. Two weeks post treatment, mice were sacrificed and RNA was extracted from cortical brain tissue and spinal cord for real-time qPCR analysis of SMN2 RNA. Results are presented as fold change in RNA levels relative to PBS control, normalized to total SMN2 levels. Each of Tables 19-23 represents a different experiment.
TABLE 19Effect of modified oligonucleotideson human SMN2 RNA splicing in homozygous transgenic miceCompoundDoseCORTEXSPINAL CORDNo.(μg)exon 7+exon 7−exon 7+exon 7...
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