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Riboswitch modulated gene therapy for retinal diseases

A switch, gene technology, applied in the field of open switches

Pending Publication Date: 2020-01-03
THE MEDICAL COLLEGE OF WISCONSIN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, toxic ligand concentrations are often necessary to "flip a switch" to turn gene expression on or off

Method used

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  • Riboswitch modulated gene therapy for retinal diseases
  • Riboswitch modulated gene therapy for retinal diseases
  • Riboswitch modulated gene therapy for retinal diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1: Ability to regulate intraocular gene expression in vivo

[0134] This example demonstrates that six riboswitches are responsive to ligands in cell culture and in vivo when delivered to the mouse retina.

[0135] Six small (~100bp) riboswitches (K19, Tc40x45, GuaM8HDV, L2Bulge18tc, L2Bulge9, and Theo6HDV) were active on ligands in cell culture, and in vivo when delivered to the mouse retina using rAAV2 vectors. response.

[0136] Using a dual-luciferase assay, riboswitches were evaluated in HEK293T cells to determine optimal copy number (maximum dynamic range) and dose-responsiveness to their activating ligands. The ligands used were tetracycline and theophylline.

[0137] Cell culture experiments showed a significant change in firefly luminescence in response to administration of the appropriate ligand (p<0.01, one-way ANOVA, N=4, all groups).

[0138] Next, optimal copy numbers of each riboswitch were cloned into rAAV GFP reporter cassettes and packaged i...

Embodiment 2

[0140] Example 2: Evaluation of optimal copy number and dynamic range of riboswitches

Embodiment 2A

[0141] Example 2A: Testing of Known Riboswitches

[0142]This example illustrates the optimal number of riboswitches that can be used in a construct and the associated dynamic range. Protocols for both assays are outlined in Figure 8A and 8B middle. To determine optimal copy number, plasmids containing 0-4 copies of each riboswitch were created, and for readout, green fluorescent protein (GFP) was used as the transgene. HEK293T cells were seeded in 12-well plates and transfected with 1 μg of each plasmid DNA on day 2. On day 3, cells were visualized by fluorescence microscopy (excitation 467-498nm) and fluorescence was quantified using a plate reader (excitation 488nm 520nm emission).

[0143] To determine the dynamic range, constructs containing firefly luciferase with an optimal copy number of riboswitches were prepared. HEK293T cells were plated on day 1 and transfected with 1 μg of plasmid DNA on day 2. Cells were treated with 0-100 [mu]M of the ligand corresponding...

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Abstract

The present invention provides constructs comprising modified riboswitches to regulate expression of a transgene within a subject. Methods of treating a disease, specifically an eye disease, are alsocontemplated.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 469,705, filed March 10, 2017, the contents of which are incorporated herein by reference in their entirety. [0003] Statement Regarding Federally Funded Research [0004] none Background technique [0005] Age-related macular degeneration (AMD) and glaucoma are the leading causes of vision loss worldwide. AMD is a common eye disease in people 50 and older. In AMD, there is damage to the macula, a small area of ​​millions of photoreceptor cells near the center of the retina and the part of the eye needed for clear central vision and the ability to see objects directly in front of you . Macular damage results from the formation of deposits called drusen and, in some cases, the growth of abnormal blood vessels under the retina. [0006] Glaucoma is a group of diseases in which the optic nerve of the eye is damaged, leading to vision loss and blin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00A61K48/00A01N25/00C12N15/63C12N15/113
CPCC12N15/67C12N2310/141C12N2310/16C12N2310/3519C12N2320/50A61K48/005A61K48/0075C12N2750/14143C12N2830/00A61P27/06C12N15/115
Inventor D·M·立平斯基C·A·里德
Owner THE MEDICAL COLLEGE OF WISCONSIN INC
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