Recombinant Hemostatic Composition via CRISPR Multiplexing

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Solution Overview

Problem

Current methods for producing coagulation therapy products, such as fresh frozen plasma and prothrombin complex concentrates, face challenges including supply chain shortages, high costs, and the need for costly pathogen screening due to reliance on pooled plasma from multiple donors.

Innovation Solution

A method for producing a wholly recombinant hemostatic composition comprising four human clotting factors (FII, FVII, FIX, and FX) using a hepatocyte cell line and CRISPR/Cas9 gene activation multiplexing, allowing for simultaneous high-level expression of these clotting factors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If pooled plasma from multiple donors is used to produce coagulation therapy products, then the supply chain is established, but supply chain shortages and pathogen screening costs increase

Engineering Contradiction:

Improvesupply stabilityVSAvoidpathogen screening cost

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The patent uses recombinant DNA technology to create copies of clotting factor genes (FII, FVII, FIX, FX) and expresses them in human hepatocyte cell lines. This produces identical functional proteins without requiring plasma from multiple donors, eliminating pathogen screening needs while ensuring reliable supply.

Inventive Principle:

Principle #26Copying

Solution Approach 2:

The patent extracts only the essential clotting factor genes from the complex plasma mixture and expresses them individually in cell lines. This separates the desired therapeutic components from the problematic plasma matrix, allowing production without donor plasma pooling and associated risks.

Inventive Principle:

Principle #2Taking out (Extraction)

2Reliability

If concentrated products like PCC are produced to minimize pathogen risk, then safety is improved, but the cost becomes prohibitive

Engineering Contradiction:

Improvepathogen safetyVSAvoidproduction cost

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent creates recombinant copies of clotting factors in cell lines, producing concentrated products without requiring expensive plasma pooling and fractionation processes. This dramatically reduces manufacturing costs while maintaining high purity and safety.

Inventive Principle:

Principle #26Copying

Solution Approach 2:

The patent uses transient transfection of hepatocyte cell lines with expression vectors, allowing single-use or limited-use cell cultures that can be rapidly produced and discarded after harvest, reducing the need for expensive, long-term GMP-certified continuous culture systems.

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Manufacturing precision

If multiple clotting factors are produced separately to ensure purity, then product uniformity is achieved, but manufacturing complexity increases

Engineering Contradiction:

Improveproduct uniformityVSAvoidmanufacturing process complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent co-transfects hepatocyte cell lines with multiple expression vectors containing different clotting factor genes (FII, FVII, FIX, FX), enabling simultaneous production of all four factors in a single cell culture system. This merges multiple manufacturing processes into one unified operation.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent uses a universal human hepatocyte cell line platform that can produce multiple different clotting factors when provided with appropriate expression vectors. This multi-functional cell system replaces the need for separate specialized production lines for each factor.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 resulting recombinant composition provides a stable, affordable, and safe source for coagulation therapy, capable of mitigating coagulopathy and reversing coagulopathic states, while being shelf-stable and non-immunogenic.

Implementation Method 1

CRISPR/Cas9 gene activation multiplexing allows for simultaneous and high level expression of the 4-FPCC constituents

Methodology Applied

Scientific EffectCRISPR-Cas9 gene targeting:

Implementation Method 2

aptamers capable to form RNA aptamer stem loops into the respective guide RNAs and which specifically bind/hybridize to the respective human clotting factor

Methodology Applied

Scientific EffectRNA hybridization:

Data Source

PatentUS20250121041A1Hemostatic composition containing recombinant human clotting factors, and method of producing

Publication Date: 2025.04.17 REGENTS OF THE UNIVERSITY OF MINNESOTA

AI summary

This invention relates to a wholly recombinant four factor hemostatic complex concentrate (4F-PCC) composition, human liver cell-produced product. Methods of producing a hemostatic composition using a hepatocyte cell line and a CRISPR/Cas9 gene activation multiplexing, which allows for simultaneous and high level expression of the FII, FVII, FIX, and FX clotting factors. The resultant product is a recombinant resuscitation solution to aid in mitigation of coagulopathy, reversal of coagulopathic states, and fluid resuscitation.