Neuropeptide-expressing vectors and methods for treatment of epilepsy

A technology for epilepsy and delivery vectors, which can be used in neurological diseases, chemical instruments and methods, biochemical equipment and methods, etc., and can solve problems such as unimproved seizure rate.

Pending Publication Date: 2019-06-04
CHARITE MEDICAL UNIV BERLIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite the large number of antiepileptic drugs introduced over the past few decades, the seizure rate (30% to 70%) o...

Method used

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  • Neuropeptide-expressing vectors and methods for treatment of epilepsy
  • Neuropeptide-expressing vectors and methods for treatment of epilepsy
  • Neuropeptide-expressing vectors and methods for treatment of epilepsy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0244] 1.1 AAV construct

[0245] The plasmid with the AAV2-ITR flanking the vector backbone contains the human CMV-IE gene enhancer, followed by the truncated form of the chicken beta actin promoter. These drive full-length codon-optimized human ppdyn sequences (AA SEQ ID No. 1) or variants (AA SEQ ID No. 2; SEQ ID No. 3) or truncated variants (□GFP) Or expression of the full-length form of EGFP. Gene expression is enhanced by the woodchuck hepatitis virus post-translational enhancer element (WPRE), followed by poly A derived from bovine growth hormone + Signal sequence. AAV2ITR is in its wild-type configuration to produce an AAV vector with ssDNA genome, or one ITR is truncated to produce a self-complementary AAV vector with dsDNA genome ( figure 1 ).

[0246] 1.2 AAV vector preparation

[0247] HEK 293 cells were seeded in DMEM containing 5% FCS at 25% to 33% confluence. After 24 hours, the cells were transfected by calcium phosphate co-transfection. The production of AAV ve...

Embodiment 2

[0253] animal

[0254] C57BL / 6N wild-type and pDyn knockout (pDyn-KO) mice were investigated in this study. The pDyn-KO mice were backcrossed to the C57BL / 6N background for 10 generations (Loacker et al., 2007). In order to breed and maintain, the mice are raised in groups and have free access to food and water. The temperature was fixed at 23° C. and 60% humidity, with a 12-hour light-dark cycle (lighting from 7 am to 7 pm). All procedures involving animals were approved by the Austrian Animal Experimentation Ethics Board in accordance with the European Convention for the Protection of Vertebrates for Experimental and Other Scientific Purposes ETS Number: 123 and the Canadian Animal Protection Committee. We make every effort to reduce the number of animals used.

Embodiment 3

[0256] Kainic acid injection and electrode implantation

[0257] Male mice (12 to 14 weeks) were sedated with ketamine (160 mg / kg, i.p.; Graeub veterinary product, Switzerland), and then deeply anesthetized with sevoflurane through a precision vaporizer (Midmark, USA). As mentioned earlier, mice were injected with 50 nl20mM KA solution into the hippocampus (RC-1.80mm; ML+1.80mm; DV-1.60mm) (Loacker et al., 2007). Two electrodes (a cortical electrode and a depth electrode) were implanted immediately after KA administration. An epoxy resin-coated tungsten depth electrode (diameter 250 μm; FHC, USA) was placed in the hippocampus, targeting the CA1 area (RC-1.80mm; ML+1.80mm; DV-1.60mm). The surface electrode is a gold-plated screw (RC+1.70mm; ML+1.6mm, bregma as a reference point) placed in the skull on top of the motor cortex to monitor the summary of abnormal EEG activity. An additional surface electrode is placed on the cerebellum as ground and reference. The electrodes were f...

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Abstract

The present invention provides delivery vectors for transferring a nucleic acid sequence to a cell in vitro, ex vivo or in vivo. The present invention provides methods of delivering a nucleic acid sequence to a cell and methods of treating focal epilepsies.

Description

Technical field [0001] The present invention provides a delivery vector for transferring a nucleic acid sequence encoding a propeptide into a cell in vitro, in vitro or in vivo. The present invention provides methods for delivering nucleic acid sequences into cells and methods for treating focal epilepsy. Background technique [0002] The prevalence of epilepsy is 1% to 2%, which is the most common neurological disease in the world (McNamara et al., 1999). Medial temporal lobe epilepsy (mTLE) is the most common type of epilepsy in humans and is often induced by traumatic brain injury. Hippocampal sclerosis and accompanying neurological deficits are key features of mTLE (for a review, see Engel et al., 2001). Despite the introduction of a large number of anti-epileptic drugs in the past few decades, the incidence of drug-resistant epilepsy (30% to 70%) has not improved since Coatsworth's study in 1971 (Coatsworth et al., 1971; Loscher et al., 2011). So far, surgical resection ...

Claims

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Application Information

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IPC IPC(8): C07K14/665C07K14/70
CPCC07K14/665C07K14/70C12N15/85C12N15/86C12N2750/14141A61P25/08A61P43/00
Inventor 雷吉娜·海尔布隆克里斯托弗·施瓦策尔
Owner CHARITE MEDICAL UNIV BERLIN
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