Gene editing systems comprising an RNA guide targeting lactate dehydrogenase a (LDHA) and uses thereof
Inventors
WESSELLS, Quinton Norman • HASWELL, Jeffrey Raymond • Ditommaso, Tia Marie • Jakimo, Noah Michael • SENGUPTA, Sejuti
Assignees
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Publication Number
US-11939607-B2
Publication Date
2024-03-26
Expiration Date
Abstract
Provided herein are gene editing systems and/or compositions comprising RNA guides targeting LDHA for use in genetic editing of the LDHA gene. Also provide herein are methods of using the gene editing system for introducing edits to the LDHA gene and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.
Core Innovation
The invention provides a gene editing system for genetic editing of a lactate dehydrogenase A (LDHA) gene. The system comprises a Cas12i2 polypeptide variant of SEQ ID NO:1166 with specified amino acid substitutions at positions comprising D581, I926, and V1030, together with an RNA guide or a nucleic acid encoding the RNA guide.
The RNA guide includes a spacer sequence specific to a target sequence within an LDHA gene. The target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the motif 5′-TTN-3′, and the PAM is located 5′ to the target sequence. The disclosure also includes defined RNA guide structure including spacer and direct repeat, and sequence listings.
The described embodiments include LDHA target locations such as exon 3 or exon 5, and delivery and use contexts including nucleic-acid and viral vector forms. The document further states therapeutic use associated with primary hyperoxaluria (PH), including PH1/PH2/PH3, and pharmaceutical composition and kit embodiments.
Claims Coverage
The independent claims cover a gene editing system for genetically editing an LDHA gene using a Cas12i2 polypeptide variant with specified mutations and an RNA guide with an LDHA-targeting spacer. The claim coverage identifies 2 inventive features: the specified Cas12i2 variant and the LDHA-targeting RNA guide with a PAM motif of 5′-TTN-3′ located 5′ to the target sequence.
Cas12i2 polypeptide variant with specified SEQ ID NO:1166 mutations
A Cas12i2 polypeptide, or a first nucleic acid encoding the Cas12i2 polypeptide, wherein the Cas12i2 polypeptide is a variant of SEQ ID NO:1166 and comprises mutations at positions comprising D581, I926, and V1030, with amino acid substitutions of D581R, I926R, and V1030G, respectively.
LDHA-targeting RNA guide with 5′-TTN-3′ PAM positioned 5′ to the target
An RNA guide, or a second nucleic acid encoding the RNA guide, wherein the RNA guide comprises a spacer sequence specific to a target sequence within an LDHA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5′-TTN-3′, which is located 5′ to the target sequence.
Across the independent claim coverage, genetic editing of an LDHA gene is implemented by combining a specified Cas12i2 polypeptide variant (relative to SEQ ID NO:1166 with D581R, I926R, V1030G substitutions) with an RNA guide whose spacer targets an LDHA sequence adjacent to a 5′-TTN-3′ PAM positioned 5′ to the target.
Stated Advantages
Smaller effector size versus SpCas9 and Cas12a.
Larger deletions.
Different PAM usage.
Potentially improved specificity versus SpCas9, including off-target identification via TTISS.
Documented Applications
Therapeutic use for primary hyperoxaluria (PH1/PH2/PH3) via LDHA reduction to address oxalate production and calcium oxalate crystal deposition.
Pharmaceutical composition and kit embodiments containing the LDHA-targeting gene-editing components.
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