Variant rnai against alpha-synuclein

A technology of synuclein and carrier, applied in the direction of DNA / RNA fragments, single-stranded DNA virus, recombinant DNA technology, etc., can solve the problem of reduction

Pending Publication Date: 2021-05-14
GENZYME CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, therapeutic strategies to reduce SNCA levels could potentially

Method used

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  • Variant rnai against alpha-synuclein
  • Variant rnai against alpha-synuclein
  • Variant rnai against alpha-synuclein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0279] Example 1: shmiRNAs reduce human SNCA targets in vitro.

[0280] To demonstrate the reduction of SNCA expression in vitro, a plasmid encoding a candidate RNAi sequence and a plasmid encoding human SNCA cDNAA (NM_000345.3) (under the control of the cytomegalovirus enhancer and hEF1a promoter / intron, and the above coding sequence Then Tbgh poly A sequence) transfected HEK293T cells. SNCA cDNA and miRNA plasmids were co-transfected.

[0281] method

[0282] siRNA and plasmids

[0283] The RNAi sequence was designed in-house to have maximum homology to human / rhesus / mouse SNCA, while it was balanced with low off-target potential. Conventional siRNAs were synthesized by Sigma. Prediction of off-target genes in the human transcriptome using the siSPOTR algorithm (World Wide Web https: / / sispotr.icts.uiowa.edu / sispotr / tools.html).

[0284] A plasmid expressing an RNAi sequence targeting SNCA was embedded within a murine mir155 scaffold driven by the cytomegalovirus enhanc...

Embodiment 2

[0291] Example 2: shmiRNAs reduce mouse SNCA targets in vitro.

[0292] A method similar to Example 1 was used in this experiment, except that HEK293T cells were transfected with plasmids encoding the indicated RNAi sequences and mouse SNCA cDNA.

[0293] result

[0294] As shown in Figure 3, the indicated miRNAs reduced mouse SNCA protein levels in vitro, with 14 / 27 sequences showing greater than 50% reduction in protein levels.

Embodiment 3

[0295] Example 3: siRNAs reduce human and mouse SNCA targets in vitro.

[0296] Additional experiments using siRNA formats were performed for A1, A2, B1, B2, C1, C2, D1, D2, E1 and E2 to provide additional data on target reduction as these siRNA formats were designed with additional features , the additional feature is having complete or significant homology to rat SNCA, which enables flexible selection of future animal models of neurodegeneration that may require rat SNCA reduction.

[0297] method

[0298] In vitro cell culture and transfection

[0299] HEK293T cells were grown to approximately 70%-90% confluency in DMEM+10% FBS+pen / step. Cells were transfected with Lipfectamine 2000 (for the miRNA format plasmid) or RNAiMAX (for the siRNA format). Three days after transfection, cells were washed with phosphate-buffered saline and whole-cell extracts were prepared by lysing cells in reduced Laemmli buffer and boiling for 5 min before storing at −20°C.

[0300] Western ...

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Abstract

Provided herein are RNAi molecules for treating neurodegenerative synucleinopathies. In some embodiments, the RNAi molecules target expression of alpha-synuclein (SNCA). Further provided herein are expression constructs, vectors ( e.g rAAV), cells, viral particles, and pharmaceutical compositions containing the RNAi. Yet further provided herein are methods and kits related to the use of the RNAi, for example, to treat neurodegenerative synucleinopathies including Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 714,616, filed August 3, 2018, which is hereby incorporated by reference in its entirety. [0003] Submission of sequence listings in ASCII text files [0004] The contents of the following submitted ASCII text file are hereby incorporated by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 159792016540SEQLIST.TXT, Date of Record: July 31, 2019, Size: 19KB) . technical field [0005] The present disclosure relates to variant RNAi molecules. In some aspects, the disclosure relates to variant RNAi to alpha-synuclein. Background technique [0006] Long-term neuroprotective therapy has considerable implications for the treatment of neurodegenerative synucleinopathies such as Parkinson's disease or multiple system atrophy (MSA). RNA interference (RNAi) is the mechanism by which target mRNA is r...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/14C12Y207/11001A61K31/7105A61P25/28C12N2750/14143C12N15/86C12N2320/32C12N2830/38C12N2830/42C12N2830/50
Inventor B·埃尔默B·理查兹M·拉塔·马希欧M·G·奥比努V·陶平V·布兰查德
Owner GENZYME CORP
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