Riboswitch modulated gene therapy for retinal diseases
Inventors
Lipinski, Daniel M. • REID, Chris A.
Assignees
Publication Number
US-12012602-B2
Publication Date
2024-06-18
Expiration Date
2038-03-09
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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 also contemplated.
Core Innovation
The present invention provides exogenous nucleic acid constructs utilizing modified riboswitches, specifically self-targeting ligand inactivating microRNA (SLIM) switches, for regulating expression of a transgene within a subject. The SLIM switches function as ON-type riboswitches, mediating increased gene expression in the presence of a ligand, with regulation achieved via a dual mechanism of gene silencing. The SLIM system combines an aptamer domain capable of binding to a ligand with a pri-miRNA sequence, allowing gene expression to be turned on or off depending on ligand presence.
The background identifies the need for effective, controllable gene therapies for ocular diseases like Age-Related Macular Degeneration (AMD) and glaucoma, because current treatments are invasive, expensive, and burdensome. Previous approaches to controllable gene expression, namely inducible promoter systems, are of limited applicability due to restricted coding capacity of viral vectors such as adeno-associated virus (AAV), preventing integration of large regulatory elements.
The innovation overcomes these limitations by providing SLIM riboswitch-based regulatory devices with a small genetic footprint that fit within vectors like rAAV. These riboswitches enable ligand-controlled and precise gene regulation within ocular gene therapy suitable for treating retinal conditions. The constructs can be tailored for specific regulatory targets, such as intermittent switching on of a VEGF inhibitor for AMD or modulation of prostaglandin synthesis transgenes for glaucoma, allowing dosing of activating ligands to control therapeutic transgene expression in the eye.
Claims Coverage
There is one independent claim that establishes the inventive construct and its dual mechanism for modulating transgene expression using a modified riboswitch with both aptamer and pri-miRNA elements.
Exogenous nucleic acid construct with dual-modality smiRNA riboswitch
The construct encodes: - a target gene, - at least one smiRNA switch selected from SEQ ID NOs: 1, 21-35, and 37-51, located within the untranslated region of the transgene; the smiRNA switch includes an aptamer domain capable of binding to a ligand and a pri-miRNA, - at least one miRNA target sequence complementary to at least a portion of the pri-miRNA. The smiRNA switch regulates transgene expression by two mechanisms: 1. Regulation of cleavage of the mRNA of the transgene. 2. Regulation of cleavage of the pri-miRNA from the smiRNA, where at least part of the cleaved pri-miRNA binds to the miRNA target sequence, silencing the transgene expression.
The inventive feature centers on an exogenous nucleic acid construct including a specially designed smiRNA riboswitch combining aptamer and pri-miRNA elements, enabling dual-mode ligand-dependent regulation of transgene expression through mRNA cleavage and miRNA-mediated silencing.
Stated Advantages
The riboswitch-based system has a small genetic footprint (~100 bp), allowing easy incorporation into viral vectors like rAAV without sacrificing coding capacity.
RNA-based devices act in cis, limiting the likelihood of an immune response since no protein cofactors are required for functionality.
The aptamer domain of the riboswitch can be engineered to respond to a wide range of activating ligands, including small molecules, proteins, or ions.
The SLIM switches provide improved control of transgene expression through a dual mechanism of gene silencing and a distinct design over prior riboswitches.
SLIM riboswitches can operate at non-toxic ligand concentrations, overcoming the limitation of prior riboswitches that required toxic ligand levels to function.
The technology allows precise, ligand-controlled, and reversible expression of therapeutic genes in the eye, enabling intermittent gene activation or silencing according to disease needs.
Documented Applications
Gene therapy for age-related macular degeneration (AMD) by regulating expression of a VEGF inhibitor transgene, such as aflibercept or sFLT1, to prevent or reduce choroidal neovascularization (CNV).
Gene therapy for glaucoma by regulating the expression of genes involved in prostaglandin synthesis (e.g., PTGS2 and PTGFR) in the eye to reduce intraocular pressure.
General regulation of therapeutic transgene expression in the retina or cornea using rAAV-based vectors with SLIM riboswitches for ocular diseases.
Use in kits providing rAAV vector constructs and doses of activating ligand for controlled transgene expression in gene therapy for eye diseases.
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