Methods of identifying genes involved in memory formation using small interfering rna (siRNA)

A memory and gene technology, applied in the field of using small interfering RNA (siRNA) to identify genes involved in memory formation, which can solve the problems of unproven specific role of RNAi, low efficacy of naked siRNA, and limitation of successful siRNA delivery.

Inactive Publication Date: 2011-04-13
HELICON THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, successful delivery of synthetic siRNAs to the CNS in vivo is limited due to the low potency of naked siRNAs, requiring the use of large amounts of siRNAs or the need for siRNAs to be expressed from viral vectors (Thakker et al., 2004, Proc. Natl Acad Sci USA101: 17270-17275); (Xia et al., 2002, Nat. Biotechnol. 20: 1006-1010)
In addition, a specific effect of RNAi on memory formation has not yet been demonstrated

Method used

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  • Methods of identifying genes involved in memory formation using small interfering rna (siRNA)
  • Methods of identifying genes involved in memory formation using small interfering rna (siRNA)
  • Methods of identifying genes involved in memory formation using small interfering rna (siRNA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 - Screening of siRNA targeting CREB and PPI using Neuro 2A cells

[0116] Screening of a panel of siRNAs targeting the α-isoform of CREB and PP1 in the Neuro2A mouse neuroblastoma cell line. Identify some suitable siRNAs that can efficiently target CREB and PP1α without affecting the mRNA levels of some control genes ( figure 1 ).

[0117] In vivo grade siSTABLE siRNA (Dharmacon Inc., Lafayette, USA). siRNAs are chemically modified to enhance stability. 21-mer siSTABLE non-targeting siRNA was used as a control (sense strand: 5'-UAGCGACUAAACACAUCAAUU-3' (SEQ ID NO: 1); antisense strand: 5'-UUGAUGUGUUUAGUCGCUAUU-3') (SEQ ID NO: 2) (Dharmacon Inc., Lafayette, USA). siRNAs were designed using a multi component rational design algorithm (Reynolds, A. et al. Nat Biotechnol 22, 326-30 (2004)).

[0118] Real-time PCR . Neuro 2A cells were treated with 100 nM siSTABLE siRNA and Dharmafect3 vector (Dharmacon). RNA was isolated using the QIAgen RNeasy kit (Qi...

Embodiment 2

[0131] Example 2 - In vivo delivery of synthetic CREB siRNA in mice

[0132] In vivo delivery of synthetic siRNAs in the CNS is hampered by limited diffusion and uptake.

[0133] Subjects Young adult (10-12 weeks old) C57BL / 6 male mice were used. Upon arrival, mice were housed in groups (5 mice) in standard laboratory cages maintained on a 12:12 hour light-dark cycle. Always perform experiments during the photoperiod. After hippocampal cannulation, mice were housed individually in individual cages and maintained until the end of the experiment. Mice had free access to food and water except for training and testing times. Mice were maintained and bred under standard conditions consistent with the guidelines of the National Institutes of Health (NIH) and approved by the Institution Animal Care and Use Committee.

[0134] Animal surgery and siRNA injection. For siRNA injection, mice were anesthetized with 20 mg / kg Avertin and a 33-gauge catheter (coordinates: A=-1.8 mm, L=+...

Embodiment 3

[0142] Example 3 - Effect of siRNA-mediated CREB knockdown on context and trace conditioning

[0143] To examine the effect of siRNA-mediated CREB knockdown on contextual fear conditioning. siRNA targeting the region common to all splice variants of the CREB gene (positions 1114-1132 of NM_009952, corresponding to exon 7 of the CREB gene) was used. Named according to Lonze and Ginty, 2002, Neuron, 35,: 605-623. Mice were treated with CREB siRNA1 or non-targeting control siRNA once a day for 3 consecutive days. Behavioral testing begins after 3 days (see also image 3 b). This design was chosen based on pilot experiments on siRNA knockdown in the hippocampus, as previous studies have shown that gene knockdown by siRNA duplexes occurs in the CNS after a few days ((Salahpour et al., 2007, Biol. Psychiatry 61: 65-69) Tan et al., 2005, Gene Therapy 12: 59-66; Thakker et al., 2004, Proc. Natl. Acad. Sci USA 101: 17270-17275).

[0144]Situational conditioning was performed ess...

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Abstract

The present invention relates to a method of identifying a gene or gene product associated with transcription dependent memory formation in an animal comprising the steps of: (a) administering to said animal sufficient small interfering RNA (siRNA) specific for the gene to inhibit gene function; (b) training said animal under conditions sufficient to induce transcription dependent memory formation in a normal untreated animal; and (c) determining the level of transcription dependent memory formation induced by the training of the treated animal. The present invention provides methods of using small interfering RNAs (siRNA) in hippocampus to identify genes and gene product whose inhibition affects contextual and temporal long-term (LTM) memory, but not short-term memory (STM).

Description

[0001] related application [0002] This application claims the benefit of U.S. Provisional Application Serial No. 60 / 938,163, filed May 15, 2007, under 35 U.S.C. $119, which is hereby incorporated by reference in its entirety and content. field of invention [0003] The present invention relates to methods of using small interfering RNA (siRNA) molecules to identify genes involved in memory formation. Background of the invention [0004] Many living organisms, including humans, have the property of remembering past events. This property has been studied for decades, and more information is now available to explain its many clades. For example, two basic types of memory have been identified: transcription-independent memory, which includes short-term memory, and transcription-dependent memory, which includes long-term memory. [0005] Identification of genes associated with memory formation provides (a) the genetic epidemiology of cognitive impairment, (b) for individuals...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/70
CPCC12N2320/12C12N15/111A61D99/00A61K31/7105C12Q1/68C12Q2525/207C12Q2600/178
Inventor E.·m.·R·斯科特R·布尔图拉德兹M·彼得斯T·P.·图利
Owner HELICON THERAPEUTICS INC
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