Novel approach for treating cancer
An oligonucleotide and nucleotide technology, which is applied in the fields of biochemical equipment and methods, pharmaceutical formulations, and medical preparations containing active ingredients, and can solve problems such as no solutions have been found.
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Embodiment 1
[0065] Example 1: Sequence selection and oligonucleotide modification methods
[0066] First, suitable sequences representing possible target sites are identified using proprietary bioinformatics tools. In a second step, computers predict the effects of chemical modifications on these sequences to optimize modification patterns.
[0067] Stepwise sequence selection method.
[0068] Sequence lengths in the range of 13-mer lengths to 17-mer lengths were considered.
[0069] In the primary analysis, the cross-reactivity of potential target sites on human PD-L1 mRNA (NM_014143.3) and cynomolgus monkey mRNA was investigated. Cross-reactivity to nonhuman primates is thought to be important for assessing the pharmacology of exaggeration. From the 16,380 sequences originally analyzed, approximately 50% showed 100% homology to cynomolgus monkey PD-L1 mRNA.
[0070] These sequences were further analyzed for specificity to spliced human and cynomolgus transcriptomes using the fol...
Embodiment 2
[0080] Example 2: In Vitro Screening of Antisense Oligonucleotides
[0081] Material:
[0082] HDLM-2 cell line-DSMZ (Deutsche Sammlung für Mikroorganismen und Zelllinien, Braunschweig, Germany / ACC-17)
[0083] Table 1. Equipment used in the screening protocol
[0084] manufacturer serial number centrifuge Heraeus Megafuge 16R 75004270 / 41826277 Laminar flow Thermo Fisher S2020 1.2 / 41820790 Heracell Vios 16oi Incubator Thermo Fisher Clariostar BMG BMG Novocyte 3000 Flow Cytometer ACEA 45-1-1405-1055-1
[0085] Table 2. Reagents used in the screening protocol
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[0087] Table 3. List of antisense oligonucleotides:
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[0092] *Neg1 (described in WO2014 / 154843A1) and S6 were used as control ASO in the experiments and did not hybridize to the human PD-L1 sequence (NM_014143.3).
[0093] Program:
[0094] The HDLM-2 cell line was purchased from DSMZ, expanded fo...
Embodiment 3
[0100] Example 3: Downregulation of PD-L1 protein expression by selected antisense oligonucleotides
[0101] To investigate the potential of oligonucleotides to downregulate targets at the protein level, we selected the five most active oligonucleotides - determined by the highest potency for target expression inhibition at the RNA level in both cell lines. (Selected oligonucleotides: A03021; A03053; A03043; A03037; A03014). Therefore, starting from 10 μM; 1 μM; 0.5 μM; 0.1 μM; for each nucleotide, different concentrations of each selected oligonucleotide and scrambled (negative) controls were used in triplicate. As a control, cells were untreated ("no oligonucleotide") as described above. All remaining wells were filled with 150 μl of medium to prevent any evaporation effects.
[0102] Therefore, HDLM-2 cells were seeded at 15,000 cells / 50 μl / well in supplemented RPMI1640 in 96 round bottom well plates. For a starting concentration of 20 μM, dilute 8 μl of the oligonuc...
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