CRISPR-Cas9 NRAS Gene Knockout for BRAF-Resistant Cancer
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Solution Overview
Problem
Current cancer treatments, particularly for BRAF inhibitor-resistant cancers, face challenges due to the lack of effective NRAS-directed agents and the development of drug resistance, leading to treatment limitations and tumor relapse.
Innovation Solution
Introducing guide RNAs complementary to variant NRAS genes and a CRISPR-associated endonuclease into cancer cells to reduce NRAS expression or activity, using CRISPR/Cas12a or Cas9 systems to cleave the NRAS gene, thereby inhibiting cancer cell proliferation and tumor growth.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional chemotherapy is used to treat advanced stage cancer, then tumor growth is inhibited, but significant adverse side-effects occur and quality of life deteriorates
Solution Approach 1:
The patent changes the therapeutic parameter from non-specific chemotherapy to highly specific CRISPR/Cas9 gene editing. By designing guide RNAs that specifically target mutant NRAS sequences, the treatment achieves precise molecular-level specificity, eliminating the need for high-dose chemotherapy and its associated adverse effects while maintaining effective tumor growth inhibition
Solution Approach 2:
The patent segments the cancer treatment approach by targeting specific mutant NRAS sequences rather than treating all cancer cells uniformly. The guide RNAs are designed to recognize and bind to specific mutant sequences (e.g., Q61K, G12D, G12V), enabling selective degradation of mutant NRAS mRNA while sparing wild-type NRAS and normal cells, thus achieving tumor inhibition without systemic toxicity
2Reliability
If BRAF inhibitors are used to treat melanoma, then tumor growth is initially controlled, but drug resistance develops and tumor relapse occurs
Solution Approach 1:
The patent applies preliminary action by using CRISPR/Cas9 to preemptively eliminate or reduce mutant NRAS expression before BRAF inhibitor resistance can fully develop. By knocking out or knocking down the NRAS mutation that drives resistance, the treatment extends the duration of BRAF inhibitor efficacy and prevents tumor relapse, addressing the limitation of finite treatment duration
Solution Approach 2:
The patent implements feedback by targeting the specific molecular mechanism (mutant NRAS) that mediates resistance to BRAF inhibitors. By detecting and eliminating the resistance-conferring mutation through CRISPR guide RNA design, the system creates a feedback loop that counteracts the development of drug resistance, thereby extending effective treatment duration
3Reliability
If CRISPR/Cas9 is used to knock out NRAS gene, then NRAS expression is reduced and treatment efficacy is enhanced, but the complexity of the treatment protocol increases
Solution Approach 1:
The patent extracts the essential functional element (guide RNA) from the complex CRISPR system and optimizes it for in vivo use. By designing synthetic guide RNAs with specific sequences that target mutant NRAS, the complex Cas9 delivery and programming process is simplified into a targeted RNA-based therapy that can be administered more readily while maintaining high treatment efficacy
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 NRAS expression or activity in cancer cells, overcoming resistance to BRAF inhibitors and enhancing treatment efficacy by selectively targeting and editing the NRAS gene, potentially leading to improved patient outcomes.
Implementation Method 1
a CRISPR-associated endonuclease, whereby the one or more gRNAs hybridize to the NRAS gene and the CRISPR-associated endonuclease cleaves the NRAS gene
Implementation Method 2
one or more gRNAs that are complementary to one or more target sequences in the NRAS gene, whereby the one or more gRNAs hybridize to the NRAS gene
Data Source
AI summary
The disclosure provides a guide RNA (gRNA) comprising a DNA-binding domain and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease protein-binding domain, wherein the DNA-binding domain is complementary to a target domain from an NRAS gene. The disclosure also provides nucleic acid sequence encoding the gRNA. The disclosure further provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a CRISPR-associated endonuclease and a guide RNA that is complementary to a target domain from an NRAS gene in the subject. Methods of treating cancer comprising administering a pharmaceutical composition comprising: a nucleic acid sequence encoding a guide RNA that is complementary to a target domain from an NRAS gene in the subject; and a nucleic acid sequence encoding a CRISPR-associated endonuclease, are also provided.