NCF1 Prime Editing Without Pseudogene Chromosomal Rearrangements
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Solution Overview
Problem
Chronic granulomatous disease (CGD) is an inherited hematologic disorder caused by mutations in the NADPH oxidase enzyme, leading to susceptibility to infections and inflammation, with current treatments like hematopoietic stem cell transplantation being risky and not universally available, and CRISPR gene editing posing safety risks due to chromosomal rearrangements near pseudogenes.
Innovation Solution
Prime editing methods and compositions are used to correct mutations in the NCF1 gene, specifically targeting the NCF1 gene flanked by pseudogenes NCF1B and NCF1C, using a prime editing guide RNA (PEgRNA) to introduce precise nucleotide edits and avoid chromosomal rearrangements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If classic CRISPR gene editing is used to correct NCF1 mutations, then the mutation can be corrected, but chromosomal rearrangements occur due to multiple double strand breaks at target sites in NCF1, NCF1B, and NCF1C
Solution Approach 1:
The patent changes the fundamental parameter of the editing mechanism from double-strand breaking (classic CRISPR-Cas9) to single-strand nicking (Cas9 nickase or Cas12f1). This parameter change allows the system to make precise edits at the NCF1 c.73_74 AGT deletion site without generating the multiple double-strand breaks that cause chromosomal rearrangements. The nicking approach creates a controlled single-strand break that guides repair through the provided template RNA, achieving precision without the harmful chromosomal effects.
Solution Approach 2:
The patent introduces template RNA as an intermediary molecule that carries the correction sequence. Instead of relying on error-prone non-homologous end joining to repair double-strand breaks, the template RNA serves as a mediator that provides the exact sequence information needed for precise correction. This intermediary approach ensures that only the intended edit occurs at the NCF1 site without causing rearrangements at the pseudogene locations.
2Reliability
If hematopoietic stem cell transplantation is used to treat CGD, then curative treatment is achieved, but risks of graft versus host disease and treatment unavailability occur
Solution Approach 1:
The patent enables the patient's own hematopoietic stem cells to correct their genetic defect through in vitro prime editing, eliminating the need for allogeneic transplantation. The self-service approach uses the patient's autologous cells that are edited ex vivo and then reinfused, thereby curing the disease without introducing foreign tissue that could cause graft versus host disease. This transforms the treatment from a donor-dependent process to a self-correcting biological process.
Solution Approach 2:
The patent performs the gene correction action before the cells are transplanted back into the patient. By conducting the prime editing procedure ex vivo on the patient's stem cells prior to reinfusion, the genetic defect is corrected in advance, ensuring that the cells returned to the patient are already functional and disease-free, thereby eliminating the need for lifelong immunosuppression or donor matching.
3Manufacturing precision
If NCF1 gene editing is performed near pseudogenes NCF1B and NCF1C, then the NCF1 mutation is corrected, but multiple double strand breaks are induced leading to safety concerns
Solution Approach 1:
The patent changes the editing mechanism from double-strand breaking to single-strand nicking, fundamentally altering the safety profile. By using Cas9 nickase or Cas12f1 to create only a single-strand break at the NCF1 target site, the system avoids generating the multiple double-strand breaks that would otherwise be induced by classic CRISPR-Cas9 at the highly similar pseudogene sites. This parameter change maintains targeted correction while dramatically improving safety.
Solution Approach 2:
The patent applies local quality by designing the editing system to be highly specific to the NCF1 gene sequence at the c.73_74 AGT deletion site, while the pseudogenes NCF1B and NCF1C remain unaffected. The prime editing system with its specific guide RNA and template RNA creates a localized correction only at the intended target, leaving the pseudogene regions intact and avoiding any editing activity at those locations.
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
The prime editing approach effectively corrects the NCF1 gene mutations, potentially providing a safer and more effective treatment for CGD by enhancing the functional activity of neutrophils, reducing infection risk, and avoiding the complications of traditional therapies.
Implementation Method 1
a spacer that is complementary to a search target sequence on a first strand of a NCF1 gene
Implementation Method 2
A prime editing complex may generate a nick in the target gene on the edit strand which is the complementary strand of the target strand
Implementation Method 3
a single stranded DNA is synthesized using an editing template of the PEgRNA as a template
Data Source
AI summary
Provided herein are compositions and methods of using prime editing systems comprising prime editors and prime editing guide RNAs for treatment of genetic disorders.