Lipid formulated DSRNA targeting the PCSK9 gene
A lipid and gene expression technology, applied in the field of dsRNA composition, can solve the problems of lowering LDLc level, total cholesterol level and LDLc cholesterol level, etc.
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Embodiment 1
[0408] The gene walking of embodiment 1.PCSK9 gene (gene walking)
[0409] siRNA design was performed for identification in two independent screens:
[0410] a) siRNA targeting human and mouse or rat PCSK9 mRNA, and
[0411] b) All reactive human siRNAs predicted to be specific for the target gene PCSK9.
[0412] The mRNA sequences of human, mouse and rat PCSK9 were used: the human sequence NM_174936.2 was used as a reference sequence throughout the siRNA selection process.
[0413] In the first step, a 19-mer extension was identified that is conserved in human and mouse and in the human and rat PCSK9 mRNA sequences, resulting in human-mouse cross-reactive siRNAs as well as human- and rat-cross-reactive siRNAs siRNA screening.
[0414] siRNAs specifically targeting human PCSK9 were identified in a second screen. All possible 19mer sequences of human PCSK9 were extracted and defined as candidate target sequences. Table 1a and Table 2a list all sequences that cross-react ...
Embodiment 2
[0432] Example 2. dsRNA synthesis
[0433] Reagent source
[0434] Where the source of a reagent is not explicitly indicated herein, the reagent can be obtained from any supplier of molecular biology reagents at a standard of quality / purity consistent with molecular biology applications.
[0435] siRNA synthesis
[0436] Using Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, Proligo Biochemie GmbH, Hamburg, Germany) was used as a solid support to generate single-stranded RNA by solid-phase synthesis on a 1 μmolar scale. RNA and RNA containing 2'-O-methyl nucleotides were produced by solid phase synthesis using the corresponding phosphoramidites and 2'-O-methylphosphoramidites (Proligo Biochemie GmbH, Hamburg, Germany), respectively. These structures were synthesized using standard nucleoside phosphoramidite chemistry (e.g., as described by Current protocols in nucleic acid chemistry, Beaucage...
Embodiment 3
[0469] Example 3. PCSK9 in HuH7, HepG2, Hela and Monkey Primary Hepatocytes siRNA screening found highly active sequences
[0470] HuH-7 cells were obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources) (Shinjuku, Japan, cat. No.: JCRB0403). at 37°C and 5% CO 2 In a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany) supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, cat.No.S0115), penicillin 100U / ml, Streptomycin 100 μg / ml (Biochrom AG, Berlin, Germany, cat.No.A2213) and Dulbecco's MEM medium (Biochrom AG, Berlin , Germany, cat.No.F0435) cultured cells. HepG2 cells and Hela cells were obtained from the American Type Culture Collection (Rockville, MD, cat.No.HB-8065), and cultured at 37°C and 5%CO2 In a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany) in an atmosphere supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany, c...
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