Riboswitch modulated gene therapy for retinal diseases
Inventors
Lipinski, Daniel M. • REID, Chris A.
Assignees
Publication Number
US-12286631-B2
Publication Date
2025-04-29
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 invention provides nucleic acid constructs comprising modified riboswitches, specifically self-targeting ligand inactivating microRNA (SLIM) switches, to regulate expression of a transgene within a subject. These constructs offer a dual mechanism for gene silencing, which enables better and more controlled regulation of gene expression in gene therapy. The design allows turning gene expression on or off in response to non-toxic concentrations of specific ligands that are able to cross the blood-retinal barrier, such as tetracycline, theophylline, and guanine.
The problem addressed is the current lack of efficient and controllable gene therapy methods for diseases such as age-related macular degeneration (AMD) and glaucoma. Existing treatments for these eye diseases are invasive, expensive, and require frequent administration, which is burdensome for patients and clinics. Moreover, traditional inducible promoters are unsuitable for adeno-associated virus (AAV) vectors due to their large size relative to AAV's limited coding capacity, making fine, reversible gene regulation within safe and practical limits challenging.
This invention introduces SLIM switches into gene therapy constructs to allow regulation of specific therapeutic transgenes. SLIM switches are incorporated into the untranslated regions of the chosen transgenes, using aptamer domains that bind to a ligand and pri-miRNA sequences. Upon ligand binding, the conformation of the switch changes, preventing cleavage by Drosha and thereby increasing gene expression. In the absence of ligand, the pri-miRNA is processed to a mature miRNA, which then acts to silence the transgene post-transcriptionally. This precise and reversible regulation of gene expression enables effective, ligand-controlled treatment of retinal diseases using viral vectors such as rAAV, with the capability for dosing as needed to achieve therapeutic benefit while minimizing risks.
Claims Coverage
The patent has one independent claim focusing on nucleic acid constructs comprising specific sequences for use in gene therapy applications.
Nucleic acid construct comprising SEQ ID NO: 52, 53, or 54
The inventive feature is a nucleic acid construct that includes at least one of SEQ ID NO: 52 (PTGS2), SEQ ID NO: 53 (PTGFR), or SEQ ID NO: 54 (PTGS2-P2A-PTGFR). These sequences encode genes involved in prostaglandin synthesis and regulation for gene therapy applications relevant to ocular diseases such as glaucoma.
Viral vector comprising the nucleic acid construct
The patent covers a viral vector containing the nucleic acid construct comprising SEQ ID NO: 52, 53, or 54. The viral vector can be an adeno-associated virus (AAV), lentivirus, adenovirus, plasmid, herpes simplex virus, baculovirus, or bacteriophage, with a specific example being an AAV or AAV2 vector for targeted delivery in gene therapy.
The main inventive features cover engineered nucleic acid constructs comprising specific gene sequences for regulating prostaglandin synthesis, and their incorporation into viral vectors for use in gene therapy, especially for eye diseases.
Stated Advantages
The constructs have a small genetic footprint (~100 bp), allowing easy incorporation into rAAV vectors without sacrificing critical coding capacity.
The RNA-based devices act in cis, minimizing the potential for immune response since no protein cofactors are required for functionality.
The aptamer domain can be engineered to respond to a wide variety of activating ligands, including proteins, small molecule drugs, or ions.
The dual mechanism for gene silencing in the modified riboswitches provides superior control of transgene expression compared to traditional systems.
The technology enables gene expression to be turned on or off using non-toxic ligand concentrations, improving clinical applicability and safety.
SLIM switches have increased dynamic range over similar riboswitches known in the art, enhancing the regulation of gene therapy.
Expression of therapeutic proteins can be precisely modulated and is reversible, allowing for intermittent or sustained treatment responsive to disease state and patient needs.
The need for repetitive invasive eye injections is reduced or eliminated, decreasing patient burden and healthcare costs.
Documented Applications
Gene therapy for age-related macular degeneration (AMD) by inducible expression of anti-VEGF proteins such as Aflibercept (Eylea) in the retina.
Gene therapy for glaucoma by regulating prostaglandin synthesis in the eye through expression of PTGS2 and PTGFR transgenes.
Use of viral vectors, specifically rAAV, containing the described constructs for targeted delivery in ocular gene therapy.
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