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Materials and methods for delivering nucleic acids to cochlear and vestibular cells

A technology of cells and inner hair cells, applied in chemical instruments and methods, biochemical equipment and methods, electrotherapy, etc.

Active Publication Date: 2019-02-05
MASSACHUSETTS EYE & EAR INFARY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been a challenge to efficiently target and transduce IHC and OHC and possibly other inner ear cells of relevance to gene therapy approaches

Method used

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  • Materials and methods for delivering nucleic acids to cochlear and vestibular cells
  • Materials and methods for delivering nucleic acids to cochlear and vestibular cells
  • Materials and methods for delivering nucleic acids to cochlear and vestibular cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1—Adeno-associated virus (AAV) containing parental AAV capsid protein leads to safe and effective cochlear genes transfer

[0096] In Example 1 the following methods and materials were used.

[0097] viral vector

[0098] CMV-driven eGFP transgenes and woodchuck hepatitis virus were prepared as previously described (Zinn et al., 2015, Cell Reports, 12:1056-68) at the Gene Transfer Vector Core in Massachusetts Eye and Ear (vector.meei.harvard.edu) AAV2 / 1, 2 / 2, 2 / 6, 2 / 8, 2 / 9 and AAV2 / Anc80L65 of the post-transcriptional regulatory element (WPRE) expression cassette. AAV2 / Anc80L65 plasmid reagents are available through addgene.com.

[0099] in vitro plant culture

[0100] For evaluation as described in an earlier publication (Dilwali et al., 2015, Scientific Reports, 5: 18599), a total of 156 cochlear explant cultures were prepared at postnatal day 4 using mouse pups of both strains. Briefly, mouse temporal bones were removed after decapitation, and the coc...

Embodiment 1A

[0105] Example 1A - Histological Analysis

[0106] After a follow-up period of 5 to 29 days, animals were sacrificed and cochlear whole-tissue embeddings were prepared according to previous reports (Sergeyenko et al., 2013, J. Neurosci., 33:13686-94). Antibodies against myosin 7A (Myo7A, #25-6790 Proteus Biosciences, Ramona, CA, 1:400) and β-tubulin (TuJ1, #MMS-435P Biolegend, SanDiego, CA, 1:200) were used, and The corresponding secondary antibodies (Alexa Fluor 555 anti-mouse antibody and AlexaFluor 647 anti-rabbit antibody, #A-21422 and #A-21245 Thermo Fisher Scientific, Waltham, MA, 1:1000) were used for cochlear whole tissue embedding Plants and explants were stained (Dilwali et al., 2015, Scientific Reports, 5:18599). Observe slides of specimens by confocal microscopy. Each image of a given experimental series was acquired using the same experimental setup, choosing laser intensity based on the specimen with the strongest eGFP signal to prevent saturation of fluoresc...

Embodiment 1B

[0108] Example 1B - Quantification of eGFP expression

[0109] For in vitro data, the cochlea was manually quantified to determine the percentage of eGFP-positive inner hair cells (IHC) and outer hair cells (OHC) by counting eGFP-positive cells in 1 or 2 100 μm sections of basal and apical samples per specimen Determining the total number of outer hair cells or inner hair cells. All visible SGNs in cochlear explants were evaluated according to their eGFP expression. The areas of the spiral border and Sertoli cells were assessed in a quantitative manner (corrected for each experimental series as indicated above) scoring from 0 (no expression) to 3 (maximum signal). A control sample without AAV was used to rule out autofluorescence. The data indicate that Anc80 targets IHC and OHC with efficiencies between 60 and 100% in the apical and basal of the two mouse lines tested. Anc80 showed consistent and qualitatively brighter IHC and OHC eGFP expression compared to AAV2 ( fig...

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Abstract

Provided herein are materials and methods for efficiently delivering nucleic acids to cochlear and vestibular cells.

Description

technical field [0001] The present disclosure generally relates to materials and methods for delivering nucleic acids to cochlear and vestibular cells. Background technique [0002] Genetic based hearing loss is an important problem for which there are few treatment options other than cochlear implants. Inherited hearing problems are usually due to a single gene defect. Prelingual deafness is diagnosed in 1 in 500 infants, and about 50% of these have a genetic cause. Usher syndrome, which accounts for 3 to 6% of deafness in young children, is associated with a number of different clinical subtypes, each of which can be caused by mutations in any of a number of different genes, whereas the TMC1 gene One genetic defect is more common, accounting for an estimated 1-2% of all inherited deafness. [0003] The inner ear (e.g., the cochlea, especially the inner and outer hair cells (IHC and OHC) in the cochlea) is used to intervene in hearing loss and deafness of various etiolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K47/42A61K48/00A61N1/32A61P27/16C07K14/005
CPCA61K48/0008A61K48/005A61K38/185A61K39/395A61P27/02A61K2039/53A61K39/00A61N1/32C07K14/005C12N15/86C12N2750/14122C12N2750/14143C12N2750/14145A61K9/0019A61K9/0046A61P25/02A61P27/16
Inventor K.斯坦科维奇L.H.范登伯格J.霍尔特G.加莱奥克
Owner MASSACHUSETTS EYE & EAR INFARY