Spinal subpial gene delivery system

A gene delivery, pial technology, applied in gene therapy, genetic engineering, muscular system diseases, etc., can solve problems such as invasiveness

Active Publication Date: 2017-12-01
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main limitation of this technique is its invasiveness, as direct spinal parenchymal needle penetration is required

Method used

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  • Spinal subpial gene delivery system
  • Spinal subpial gene delivery system
  • Spinal subpial gene delivery system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Materials and methods

[0090] Animals and general surgical preparation - use adult Sprague-Dawley rats (male and female, 250-350 g; n = 16) or cross-bred from Minnesota and Göttingen strains Adult minipigs (both sexes; 30-40 kg; n=6). Rats were anesthetized with 5% isoflurane and maintained at 2-3% isoflurane during surgery depending on respiration rate and paw pinch response. Then, the hair on the rat's back was shaved and washed with 2% chlorhexidine. Following skin incision, the paraspinal muscles surrounding the cervical, thoracic, or lumbar spine were removed, and the animal was secured into a spinal cord fixation stand (Stoelting) using a Cunningham spinal cord clamp as previously described (Kakinohana et al. People (2004). Region-specific cell grafting into cervical and lumbar spinal cord in rat: aqualitative and quantitative stereological study. Experimental neurology 190: 122-132). To expose the spinal cord, a dorsal laminectomy of the corresponding vertebr...

Embodiment 2

[0098] Substantial AAV9-mediated transgene expression following a single subpial bolus

[0099] Initially, the efficacy of a single bolus subpial delivery of AAV9-UBI-GFP or AAV9-UBI-RFP was tested in rats and pigs. Animals received 20 μl (rats) or 200 μl (pig) AAV9 vector. At 6-8 weeks after AAV9 delivery, spinal cords were dissected from 4% paraformaldehyde-fixed animals and imaged in situ using the Avis fluorescence system. Transverse or horizontal spinal cord sections were then dissected from AAV9 injected segments and analyzed for the presence of GFP or RFP and co-stained with neuronal (NeuN) and glial (GFAP) antibodies. In rat and porcine spinal cords, strong GFP or RFP expression was found on the surface of the spinal cord and was easily identified by visual inspection as yellow-green or red areas. Figure 1H and 1J The picture shows the same as the blank control spinal cord ( Figure 1I ) compared to that in the transected porcine spinal cord and in the anterior r...

Embodiment 3

[0104] GFP expression in distal spinal cord segments

[0105] Next, the descending spinal tract GFP in the lumbar spinal cord was characterized following subpial injection of AAV9-UBI-GFP into the subpial space of the midthoracic (Th6-7) or lower cervical segments in rats and pigs degree of expression. Strong GFP expression was found throughout the entire lumbar spinal cord at 3-6 weeks after subpial AAV9 delivery. Using transverse lumbar (L2-L6) spinal cord sections taken from pigs, high-intensity GFP expression in transected axons in the lateral and ventral cords was easily identified without additional GFP immunostaining ( Figure 4A , a white asterisk). In these regions, similar densities of GFP+ axons were found throughout the white matter. Relatively lower numbers of GFP+ axons were found in the dorsal cord compared to the lateral and ventral cord ( Figure 4A , DF). Consistent with the extent of axonal labeling found in the white matter of the lateral and ventral c...

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Abstract

Delivery devices, systems, and methods related thereto may be used in humans for spinal delivery of cells, drugs or vectors. Thus, the system enables subpial delivery, which leads to a near complete spinal parenchymal AAV9-mediated gene expression or ASO distribution in both white and grey matter.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 110,340, filed January 30, 2015, in the United States Patent and Trademark Office, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates generally to gene therapy, and more particularly to a method and system for delivering genes and oligonucleotides into the subpial space of a mammal for transparenchymal infection of the spinal cord. Background technique [0004] Current methods used to deliver vectors or antisense oligonucleotides (ASOs) into the spinal cord parenchyma involve two techniques, each of which has substantial limitations compared to the present invention. [0005] First, intrathecal delivery was used when injecting vehicle or ASO into the intrathecal space of the spinal cord (ie, extrapial). Using this approach, no deep parenchymal transgene expression was se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61M25/06A61K35/76C12N15/113
CPCA61K35/00A61B17/3401C12N15/111C12N2310/11C12N2320/32C12N2330/51C12N15/86C12N2750/14143A61P21/00A61P21/02A61P25/00A61P25/02A61P25/14A61P25/16A61P25/20A61P25/28A61P43/00A61K9/0085A61K47/36A61K48/0075A61L29/041A61M25/0662A61M2210/1003C12N7/00
Inventor 马丁.马尔萨拉宮之原厚司
Owner RGT UNIV OF CALIFORNIA
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