Individualized CRISPR/Cas Complex Purification for B-Cell Lymphoma
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Solution Overview
Problem
Current cancer therapies struggle to selectively target cancerous or pre-cancerous cells while minimizing harm to healthy cells, leading to poor response rates and severe side effects due to tumor heterogeneity and immune system suppression.
Innovation Solution
Development of individualized CRISPR/Cas complexes tailored to specific mutations in tumor driver genes, such as PAX5 and MYC, using guide RNAs (crRNA and tracrRNA) to induce targeted cell death in cancerous or pre-cancerous cells without affecting healthy cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR/Cas complexes are produced in human cell lines (HEK 293 or HEK 293T), then high productivity and ease of manufacture are achieved, but mycoplasma contamination risk increases
Solution Approach 1:
The patent introduces an intermediary purification system consisting of affinity tags (FLAG, HA, Myc) and corresponding affinity purification matrices. This intermediary layer separates the CRISPR/Cas complex from contaminated cell components, allowing selective isolation of the therapeutic complex while removing mycoplasma and other cellular contaminants through affinity-based purification steps
2Manufacturing precision
If CRISPR/Cas complexes are purified to high homogeneity, then product quality and safety are improved, but manufacturing complexity and time increase
Solution Approach 1:
The purification process is segmented into distinct modular steps: initial affinity purification using tagged complexes, followed by size-exclusion chromatography, and final formulation. Each step targets specific contaminants and can be independently optimized, making the overall complex purification process manageable and scalable
Solution Approach 2:
Affinity tags serve as intermediary handles that enable selective purification. The tags (FLAG, HA, Myc) are temporary intermediaries that facilitate high-purity isolation through affinity matrices, then can be removed or retained depending on the final product requirements, simplifying the path to homogeneity
3Stability of the object's composition
If CRISPR/Cas complexes are formulated for stability, then storage and transport reliability are improved, but bioactivity may be compromised
Solution Approach 1:
The patent systematically optimizes formulation parameters including pH, ionic strength, buffer composition, and excipient types to identify conditions that simultaneously maintain complex stability and preserve RNA-guided DNA target recognition activity. Stability-inducing parameters are tuned to the minimum effective levels that prevent aggregation while avoiding conditions that might interfere with bioactivity
Data Source
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AI summary
The present invention relates to a method for the manufacture of individualized CRISPR/Cas complexes comprising the steps a) identifying in a tumor specimen of a human cancer patient at least one of the following cancer specific mutations: mutation at position 127736999 on chromosome 8, mutation at position 37020625 on chromosome 9 and mutation at position 36840626 on chromosome 9; b) preparing for the at least one mutation identified in a), preferably for two or all mutations identified in a), an individualized CRISPR/Cas complex, wherein each individualized CRISPR/Cas complex comprises a guide RNA and a Cas endonuclease, wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 127736999 on chromosome 8 comprises a crRNA according to SEQ ID NO 37 and a tracrRNA, wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 37020625 on chromosome 9 comprises a crRNA according to SEQ ID NO 38 and a tracrRNA, and wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 36840626 on chromosome 9 comprises a crRNA according to SEQ ID NO 39 and a tracrRNA; a method, preferably an in-vitro method, for inducing cell death in cancerous or pre-cancerous cells of B-cell-lymphocytes, a composition related thereto and individualized CRISPR/Cas complexes preferably for use in the treatment of B-cell-lymphoma or for inducing cell death in cancerous or pre-cancerous B-cell-lymphocytes.