HPRT-Deficient Stem Cell Chemoselection Without Radiation

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for hematopoietic stem cell transplantation (HSCT) face challenges in achieving efficient gene transfer and engraftment due to toxicity and insufficient selection efficiency, particularly with existing in vivo selection strategies using drug resistance genes, which pose risks of toxicity and selective disadvantages.

Innovation Solution

A radiation-free HSCT method using HPRT-deficient donor cells and 6-thioguanine (6TG) for both conditioning and chemoselection, allowing for high engraftment of genetically modified cells with reduced toxicity by exploiting the resistance of HPRT-deficient cells to 6TG's cytotoxic effects.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If in vivo selection strategies using drug resistance genes (DHFR, MDR1) are employed to enhance engraftment of gene-modified HSC, then selection efficiency is improved, but toxicity increases

Engineering Contradiction:

Improveengraftment efficiencyVSAvoidtoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent changes the selection mechanism from drug resistance genes to HPRT deficiency, which alters the biochemical parameter used for selection. HPRT-deficient cells are selected using 6-thioguanine, exploiting the fact that HPRT is required for the activation of 6-thioguanine cytotoxicity. This parameter change enables selection without the toxicity associated with conventional drug resistance genes.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If mutant forms of MGMT are used to confer chemoprotection against BCNU or temozolomide, then chemoselection capability is improved, but toxicity increases and selective disadvantage occurs at high expression levels

Engineering Contradiction:

Improvechemoselection capabilityVSAvoidtoxicity and selective disadvantage

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

Instead of using mutant MGMT to confer resistance to alkylating agents, the patent inverts the approach by using HPRT deficiency to confer resistance to 6-thioguanine cytotoxicity. The selection pressure is applied through 6-thioguanine administration, which selectively kills HPRT-wild-type cells while sparing HPRT-deficient cells. This inversion of the resistance mechanism resolves the toxicity and selective disadvantage problems.

Inventive Principle:

Principle #13The other way round (Inversion)

3Productivity

If high doses of 6TG are administered over long time periods for in vivo chemoselection, then selection efficiency is improved, but toxicity increases

Engineering Contradiction:

Improveselection efficiencyVSAvoidtoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent converts the harmful cytotoxic effect of 6-thioguanine into a beneficial selection mechanism. By exploiting the fact that HPRT-deficient cells are resistant to 6-thioguanine cytotoxicity, the patent uses the drug's harmful effect to selectively kill HPRT-wild-type cells while preserving HPRT-deficient donor cells. This transforms the potential harm into a useful selection pressure.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

4Productivity

If radiation is used for preconditioning prior to in vivo chemoselection, then myeloablation is achieved, but radiation-induced toxicity occurs

Engineering Contradiction:

Improvemyeloablation efficiencyVSAvoidradiation-induced toxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent replaces the mechanical/radiation-based myeloablation system with a biochemical system using 6-thioguanine. Instead of using radiation to achieve myeloablation, the patent uses 6-thioguanine administration to selectively kill HPRT-wild-type cells. This substitution eliminates radiation-induced toxicity while maintaining myeloablation efficiency through biochemical means.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 achieves over 75% genetically modified hematopoietic cells with low overall toxicity, enabling long-term reconstitution of bone marrow and improving the efficiency and safety of HSCT for treating various diseases.

Implementation Method 1

exploiting the resistance of HPRT-deficient cells to 6TG's cytotoxic effects

Methodology Applied

Scientific EffectEnzyme deficiency resistance: Enzyme

Implementation Method 2

administering to the subject about 1 to 5 mg/kg of the purine base analog every two to four days for two to eight weeks following the engrafting step

Methodology Applied

Scientific EffectPurine analog cytotoxicity: Chemical Bonding

Data Source

PatentUS11607427B2Method for chemoselection

Publication Date: 2023.03.21 RGT UNIV OF CALIFORNIA

AI summary

A method of radiation-free hematopoietic stem cell (HSC) transplantation comprises administering to a mammalian subject one or two doses of 2 to 10 mg/kg body weight of a purine base analog, such as 6TG as a pre-conditioning step. The method further comprises engrafting into the subject hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient donor HSCs within 48 to 72 hours of the pre-conditioning step; and administering to the subject about 1 to 5 mg/kg of the purine base analog every two to four days for two to eight weeks following the engrafting step. The method is performed in the absence of pre-conditioning via radiation. The subject is therefore not treated with myeloablative radiation in preparation for transplantation, and thus the subject is free of myeloablative radiation-induced toxicity.