Oligoribonucleotides and methods of use thereof for treatment of alopecia, acute renal failure and other diseases
A ribonucleotide, disease technology, applied in the direction of sugar derivatives, organic chemistry, carbohydrate active ingredients, etc., can solve the problem of no satisfactory treatment, no satisfactory treatment p53 polypeptide and other problems
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Embodiment 1
[0134] Example 1: Sequences that Produce Active siRNA Compounds
[0135] Using a dedicated algorithm and the known sequence of the gene p53 (SEQ ID NO: 1), the sequences of many possible siRNAs were generated. Table A presents 23 siRNAs that have been selected, chemically synthesized and tested for activity so far (see Example 2). All siRNAs are 19-mer.
[0136] Table A
[0137]
[0138] Note that in Table A above, the sense strands of siRNAs 1-23 have SEQ ID NOs: 3-25, respectively, and the antisense strands of siRNAs 1-23 have SEQ ID NOs: 26-48, respectively. siRNA compound No 1 (SEQID NO: 3 and 26) is known from literature (Dira and cBernards, Reversal of senescence inmouse fibroblasts through lentiviral suppression of p53, J.Biol.Chem. (2003) 278: 11731), siRNA No 2 (SEQ ID NOs: 4 and 27) are also known from the literature (Brummelkamp et al. Science 2002, 296:550-553). However, the use of these compounds for the methods of treatment described herein has not been pr...
Embodiment 2
[0152] Example 2: Testing the anti-p53 activity of siRNA compounds
[0153] Experimental program
[0154] I. Preparation of siRNA (double-stranded oligonucleotide)
[0155] Lyophilized oligonucleotides were dissolved in RNase-free distilled water to give a final concentration of 100 uM. The dissolved oligonucleotides were kept at room temperature for 15 min and then quickly frozen in liquid nitrogen.
[0156] Oligonucleotides were stored at -80°C and diluted in PBS before use.
[0157] II. siRNA transfection in human cells with Lipofectamine2000 reagent:
[0158] Will 2x10 5 p53-wt HCT116 or SW480 cells were seeded into 6-well plates per well. After 24 h, cells were transfected with p53 oligonucleotides with Lipofectamine2000 reagent (obtained from Invitrogen).
[0159] Do the following steps:
[0160] 1. Before transfection, replace the cell culture medium with 1500ul fresh culture medium without antibiotics.
[0161]2. In a sterile plastic tube, add Lipofectamine200...
Embodiment 3
[0188] Example 3: Model System of Alopecia
[0189] Testing active siRNA can be done on the following systems:
[0190] a. Hair loss model in mice
[0191] b. Human hair follicles cultured in vitro
[0192] c. Human hair follicles grafted to nude mice
[0193] NOTE: A system for testing active siRNAs is described in Botcharev et al., 2000, p53 is essential for Chemotherapy-induced Hair Loss, Cancer Research, 60 , 5002-5006.
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