HAO1 Gene Editing to Disrupt the Peroxisomal SKI Motif
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Solution Overview
Problem
There is no approved therapeutic treatment for primary hyperoxaluria Type 1 (PH1), a rare autosomal recessive disorder causing excessive urinary excretion of oxalate leading to kidney stones and nephrocalcinosis, despite early conservative treatments and combined liver-kidney transplantation being the best outcomes for chronic kidney disease.
Innovation Solution
Engineered nucleases, specifically targeting exon 8 of the HAO1 gene, are used to disrupt the peroxisomal targeting signal (SKI motif) of the HAO1 protein, preventing its localization to the peroxisome and reducing oxalate production by maintaining its activity in the cytoplasm, where it can be eliminated through urine.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the HAO1 protein is localized to the peroxisome through the SKI motif, then glyoxylate is efficiently converted to glycolate, but oxalate accumulates causing PH1 disease
Solution Approach 1:
The invention extracts or removes the SKI peroxisomal targeting signal motif from the HAO1 protein sequence. By deleting or mutating this specific motif, the HAO1 enzyme is prevented from localizing to the peroxisome, thereby changing its subcellular distribution to the cytoplasm where it cannot generate oxalate, thus eliminating the harmful effect while preserving enzymatic activity
Solution Approach 2:
The invention modifies the local quality of the HAO1 protein by altering its subcellular localization properties. Through targeted mutation of the SKI motif, the protein's destination is changed from peroxisomal to cytoplasmic, creating a localized functional difference that prevents oxalate formation while maintaining the enzyme's catalytic capability in the new location
2Reliability
If engineered nucleases are used to disrupt the SKI motif, then peroxisomal localization is prevented, but gene editing complexity increases
Solution Approach 1:
The invention employs engineered nucleases (such as CRISPR-Cas9, TALENs, or zinc finger nucleases) as intermediary tools to achieve precise modification of the HAO1 gene. These nucleases act as mediators that recognize specific DNA sequences and introduce targeted mutations to disrupt the SKI motif, enabling controlled genetic modification without requiring complex manual manipulation of the genome
Solution Approach 2:
The invention replaces traditional mechanical or chemical gene editing methods with enzymatic mechanisms. Engineered nucleases use programmable DNA recognition and site-specific cleavage followed by cellular repair mechanisms to achieve precise genetic modification, substituting complex physical manipulation with a more precise and controllable biochemical system
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach effectively reduces oxalate levels by disrupting the HAO1 protein's localization, thereby alleviating the symptoms of PH1 through in vitro and in vivo studies.
Implementation Method 1
engineered nucleases, specifically targeting exon 8 of the HAO1 gene, are used to disrupt the peroxisomal targeting signal (SKI motif) of the HAO1 protein
Implementation Method 2
Hydroxyacid oxidase 1 (HAO1), which is also referred to as glycolate oxidase, is the enzyme responsible for converting glycolate to glyoxylate in the mitochondrial/peroxisomal glycine metabolism pathway
Implementation Method 3
AGT is responsible for conversion of glyoxylate to glycine in the liver
Implementation Method 4
When AGXT is incapable of converting glyoxylate to glycine, excess glyoxylate is converted in the cytoplasm to oxalate by lactate dehydrogenase (LDHA)
Data Source
AI summary
Disclosed are engineered nucleases that bind and cleave a recognition sequence within a hydroxyacid oxidase 1 (HAO1) gene. The present invention also encompasses methods of using such engineered nucleases to make genetically-modified cells. Further, the invention encompasses pharmaceutical compositions comprising engineered nuclease proteins or nucleic acids encoding engineered nucleases of the invention, and the use of such compositions for treatment of primary hyperoxaluria type I.