RNAi reagents, compositions, and methods of use thereof for treating transthyretin (ttr)-related diseases
A technology of thyroxine and carrier protein, applied in biochemical equipment and methods, DNA/RNA fragments, drug combinations, etc., can solve problems such as unstable tetramer structure
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example 1
[0586]Example 1: Inhibition of TTR with TTR-GalNAc conjugates
[0587] A single dose of the TTR RNAi agent AD-43527 was administered subcutaneously to mice, and TTR mRNA levels were determined 72 hours after administration.
[0588] The mouse / rat cross-reactive GalNAc conjugate AD-43527 was selected for in vivo assessment of TTR mRNA silencing in liver in WT C57BL / 6 mice. The sequence of each chain of AD-43527 is shown below.
[0589] Strand: s = sense strand; as = antisense strand
[0590]
[0591] The ligand used was GalNAc 3 :
[0592]
[0593] This GalNAc3 ligand binds to the 3' end of the sense strand using the linker and tether shown below:
[0594]
[0595] GalNAc produced 3 The structure of the bound sense strand is shown in the following structural formula:
[0596]
[0597] Additional RNAi agents targeting TTR and having the following sequences and modifications were synthesized and analyzed.
[0598] Mouse / Rat Cross-Reactive TTR RNAi Reagents
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example 2
[0607] Section 1.01 Example 2: TTR inhibition with TTR-GalNAc conjugates is durable
[0608] Mice were administered a subcutaneous dose (either 7.5 mg / kg or 30.0 mg / kg) of AD-43527, a GalNAc-binding RNAi agent targeting TTR. TTR mRNA levels in liver were assessed at 1, 3, 5, 7, 10, 13, 15, 19, 26, 33, and 41 days after treatment using the method described in Example 1.
[0609] The results are shown in figure 2 middle. On day 19, administration of 30.0 mg / kg GalNAc-conjugated RNAi agent still demonstrated approximately 50% silencing. Complete recovery of expression occurred on day 41.
[0610] These results show that the inhibition provided by GalNAc-bound siRNA targeting TTR was durable for up to 3, 5, 7, 10, 13, 15, 19, 26 or 33 days post treatment.
example 3
[0611] Example 3. RNA synthesis and double annealing
[0612] 1. Oligonucleotide Synthesis
[0613] Oligonucleotides were synthesized on an AKTAoligopilot synthesizer or an ABI 394 synthesizer. Unless otherwise specified, a commercially available controlled microporous glass solid support (dT-CPG, 500 , PrimeSynthesis) and RNA phosphoramidites with standard protecting groups 5′-O-dimethoxytrityl N6-benzoyl-2′-tert-butyldimethylsilyl-adenosine -3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-tert-butyldi Methylsilyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N2--isobutyl Acyl-2'-tert-butyldimethylsilyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite and 5'-O-dimethoxytris Benzyl-2'-tert-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) ) for oligonucleotide synthesis. 2'-F phosphoramidite 5'-O-dimethoxytrityl-N4-acetyl-2'-f...
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