Antisense oligonucleotides targeting alpha-synuclein and uses thereof
A technology of antisense oligonucleotide and synuclein, which is used in medical preparations containing active ingredients, anti-animal/human immunoglobulins, peptide sources, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0319] Example 1: Construction of ASO
[0320] The antisense oligonucleotides described herein are designed to target multiple regions in the SNCA pre-mRNA. see Figure 1A is the genome SNCA sequence, Figure 1B is the SNCA cDNA. For example, constructing an ASO to target using such as figure 2 Regions indicated by the pre-mRNA start and pre-mRNA stop sites of NG_011851.1 and / or the mRNA start and stop sites of its mRNA are indicated. exist figure 2 and image 3Exemplary sequences (eg, SEQ ID numbers) for ASOs are described in . In some embodiments, ASOs are designed as gapmers (eg, alternating gapmers). See DES number.
[0321] figure 2 and image 3 Non-limiting examples of ASO designs for selected sequences are shown. The same method can be applied to any other sequence disclosed herein. The gapmers were constructed to contain locked nucleic acid - LNA (capital letters). For example, gapmers can have β-deoxy LNAs at the 5' and 3' ends with a phosphorothioate bac...
Embodiment 2A
[0323] Example 2A: High Assay to Measure SNCA Protein Reduction in Primary Neurons
[0324] The ability of SNCA-targeted ASOs to reduce SNCA protein expression was tested in primary mouse neurons. From PAC-Tg (SNCA A53T ) + / + ;SNCA - / - Primary neuronal cultures were established from the forebrain of ("PAC-A53T") mice carrying the complete human SNCA gene with the A53T mutation in a mouse SNCA knockout background. See Kuo Y et al., Hum Mol Genet., 19:1633-50 (2010). All procedures involving mice were performed according to animal testing methods (ATM) approved by the Bristol-Myers Squibb Animal Care and Use Committee (ACUC). Primary neurons were generated by papain digestion according to the manufacturer's protocol (Worthington Biochemical Corporation, LK0031050). Isolated neurons were washed and resuspended in Neurobasal medium supplemented with B27 (Gibco), 1.25 μM Glutamax (Gibco), 100 units / ml penicillin, 100 μg / ml streptomycin, and 25 μg / ml amphotericin B ( NBM, Invi...
Embodiment 2B
[0328] Example 2B: Spontaneous Calcium Oscillation Measurements
[0329] Reduced oscillations in intracellular free calcium concentrations (calcium oscillations) correspond to increased neurotoxicity and, therefore, may indicate reduced in vivo tolerance. To measure spontaneous calcium oscillations in primary cortical neurons, rat primary cortical neurons were prepared from Sprague-Dawley rat embryos (E19). Briefly, cerebral cortex was dissected and incubated in papain / DNase / Earle's Balanced Salt Solution (EBSS) solution at 37°C for 30-45 minutes. After triturating and centrifuging the cell pellet, the reaction was stopped by incubation with EBSS containing protease inhibitors, bovine serum albumin (BSA) and DNase. Cells were then triturated and washed with Neurobasal (NB, Invitrogen) supplemented with 2% B-27, 100 μg / ml penicillin, 85 μg / ml streptomycin and 0.5 mM glutamine.
[0330] Cells were plated at a concentration of 25,000 cells / well in 384-well poly-D-lysine-coated ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More