PAH Gene Editing With Cas9 Nickase and Reverse Transcriptase
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Solution Overview
Problem
Current methods for integrating longer sequences into a genome, such as the PAH gene, are inefficient and lack site specificity, and existing treatments for phenylketonuria (PKU) do not effectively address the genetic drivers of the condition.
Innovation Solution
A gene modifying system comprising a nucleic acid encoding a gene modifying polypeptide with a reverse transcriptase domain and Cas9 nickase, along with a template RNA, is used to target and correct mutations in the PAH gene, enabling insertion, deletion, or substitution of specific sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 is used for genome editing, then small edits can be achieved, but integration of longer sequences is less effective
Solution Approach 1:
The patent combines CRISPR/Cas9 technology with viral vectors (AAV, lentivirus, retrovirus) to merge the precision of genome targeting with the high integration efficiency of viral delivery systems. This hybrid approach allows for both precise small edits and effective integration of longer sequences by utilizing the viral vector's ability to carry and integrate larger genetic payloads at specific genomic loci targeted by CRISPR/Cas9.
Solution Approach 2:
The patent introduces viral vectors as intermediary carriers that bridge the gap between CRISPR/Cas9 targeting capability and the need for efficient long sequence integration. The viral vector serves as a mediator that delivers the therapeutic gene or sequence of interest to the CRISPR-defined target site, enabling both precision and high integration efficiency simultaneously.
2Manufacturing precision
If Cre/loxP system is used for sequence insertion, then site specificity can be achieved, but a two-step process is required
Solution Approach 1:
The patent segments the genome editing function into modular components: CRISPR/Cas9 for precise target site selection, viral vectors for sequence delivery, and homology-directed repair (HDR) templates for accurate integration. This segmentation allows each component to perform its specialized function efficiently, eliminating the need for preliminary loxP site insertion and subsequent Cre-mediated recombination.
Solution Approach 2:
The patent performs preliminary action by using CRISPR/Cas9 to pre-establish the exact target site and create appropriate DNA breaks before delivering the therapeutic sequence via viral vector. The HDR template is also prepared in advance with homology arms matching the target site, ensuring that integration occurs precisely at the desired location in a single coordinated step rather than requiring sequential operations.
3Productivity
If natural nucleic acid integration occurs, then genome integration can happen, but frequency is low and site specificity is minimal
Solution Approach 1:
The patent implements feedback control through the CRISPR guide RNA system, which uses base-pairing complementarity to provide real-time feedback on target site recognition. The gRNA sequence is designed to match the specific genomic locus, ensuring that integration only occurs at the correct site. The Cas9 nuclease activity is also regulated by gRNA binding, creating a feedback mechanism that enhances both site specificity and integration frequency by concentrating editing activity at the intended target.
Solution Approach 2:
The patent changes key parameters of the integration process by using viral vectors to dramatically increase the concentration and delivery efficiency of the therapeutic nucleic acid to the target site. The viral vector system transforms the low-frequency natural integration process into a high-efficiency event by providing forced entry into the nucleus and direct delivery to chromatin, while CRISPR/Cas9 simultaneously changes the specificity parameter by creating discrete DNA breaks only at the targeted locus.
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 system allows for precise modification of the PAH gene, potentially correcting mutations and restoring enzyme function, thereby reducing phenylalanine levels in the blood and alleviating symptoms of PKU.
Implementation Method 1
a reverse transcriptase domain
Implementation Method 2
a Cas9 nickase that binds DNA and has endonuclease activity
Implementation Method 3
a gRNA scaffold that binds the polypeptide
Data Source
AI summary
The disclosure provides, e.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject. Gene modifying systems for treating phenylketonuria (PKU) are described.