BEST1 DNA Vectors With shRNA for Safe Retinal Gene Delivery
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Solution Overview
Problem
There is a lack of safe and effective treatments for BEST1-associated retinal dystrophies, which are chronic and progressive disorders caused by BEST1 mutations affecting Ca2+ signaling and homeostasis in retinal cells, due to complex biological mechanisms and restricted access to the retina.
Innovation Solution
Nucleic acid vectors, specifically DNA vectors, are developed to modulate BEST1 expression in ocular cells by encoding a BEST1 RNA transcript and a short hairpin RNA (shRNA) that do not target the BEST1 RNA transcript, using non-viral compositions and electrotransfer for delivery to retinal cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors are used to deliver BEST1 to retinal cells, then expression efficiency is improved, but safety concerns worsen due to immunogenicity and insertional mutagenesis risks
Solution Approach 1:
The patent converts the harmful effects of viral vectors (immunogenicity, insertional mutagenesis) into beneficial features by using non-viral DNA vectors that avoid these risks while maintaining sustained expression. The non-viral approach eliminates immune responses and random integration, yet achieves durable BEST1 expression through episomal maintenance and regulated promoters.
Solution Approach 2:
The patent changes key parameters of the delivery system by transitioning from viral to non-viral vectors, modifying the mechanism of delivery (electroporation instead of viral infection), and altering expression regulation (tetracycline-inducible systems). These parameter changes maintain expression efficiency while eliminating safety hazards associated with viral vectors.
2Object-affected harmful factors
If non-viral DNA vectors are used to deliver BEST1, then safety is improved by avoiding viral immunogenicity, but delivery efficiency and cellular uptake worsen
Solution Approach 1:
The patent introduces electroporation as an intermediary physical method to facilitate DNA vector entry into retinal cells. This intermediary approach overcomes the poor cellular uptake of non-viral vectors by using controlled electrical fields to create temporary pores in cell membranes, enabling efficient DNA delivery without viral components.
Solution Approach 2:
The patent replaces the biological mechanism of viral infection with a physical-electrical mechanism (electroporation). Instead of relying on viral particles to mediate cellular entry, the system uses controlled electrical pulses to directly facilitate DNA uptake, substituting a mechanical/physical process for a biological one to improve delivery efficiency while maintaining safety.
3Measurement precision
If shRNA is included to knockdown mutant BEST1, then specificity is improved, but off-target effects and toxicity worsen
Solution Approach 1:
The patent applies local quality by designing shRNA sequences with high specificity to target only mutant BEST1 alleles. Through careful sequence selection and validation, the shRNA is engineered to recognize unique mutations while avoiding homology with wild-type BEST1 and other genomic sequences, thereby achieving localized specific action at the molecular level.
Solution Approach 2:
The patent implements preliminary anti-action by using shRNA to preemptively knockdown mutant BEST1 transcripts before they can cause pathological effects. The shRNA is designed to bind specifically to mutant transcripts and induce their degradation, preventing the formation of toxic proteins while sparing wild-type alleles, thus counteracting the harmful effects before they manifest.
4Adaptability or versatility
If multiple genetic elements are included in the vector for regulated expression, then expression control is improved, but vector complexity and manufacturing difficulty worsen
Solution Approach 1:
The patent applies segmentation by dividing the vector into distinct functional modules: promoter region, BEST1 coding sequence, shRNA sequence, and regulatory elements. This modular design allows for independent optimization and assembly of each component, facilitating standardized manufacturing while maintaining sophisticated expression control through the coordinated function of separate elements.
Data Source
AI summary
Provided herein are expression constructs, nucleic acid vectors, pharmaceutical compositions, and methods for improved expression of functional BEST1 in subjects having mutations in BEST1 and associated retinal dystrophies. Improved expression constructs include BEST 1-targeted short hairpin RNAs (shRNAs), BEST1 coding sequences, promoters driving expression of shRNAs and coding sequences, and/or additional regulatory elements to improve BEST1 expression. Expression constructs, nucleic acid vectors and pharmaceutical compositions thereof, and methods of use thereof, can provide effective treatments for BEST1-associated disorders, such as autosomal dominant bestrophinopathies.