Transposon-Mediated GBA1 Integration in Pluripotent Stem Cells

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Solution Overview

Problem

Current methods for differentiating pluripotent stem cells into lineage-specific cell populations, such as dopamine neurons, face limitations in producing cells with consistent physiological characteristics and engraftment capabilities, particularly in cases involving reduced GBA1 activity associated with neurodegenerative diseases like Parkinson's Disease.

Innovation Solution

The introduction of a DNA sequence encoding GBA1 operably linked to a promoter, positioned between inverted terminal repeats, and a transposase into pluripotent stem cells to integrate into the genome, thereby increasing GBA1 expression and GCase activity, followed by differentiation into specific neural cell types.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional differentiation methods are used to generate dopamine neurons from pluripotent stem cells, then cell production is achieved, but the cells lack consistent physiological characteristics and engraftment capability

Engineering Contradiction:

Improvephysiological consistencyVSAvoidcell production efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent applies preliminary action by integrating the GBA1 transgene into the pluripotent stem cell genome before differentiation. This ensures that GCase activity is enhanced from the earliest stages of cell development, leading to more consistent physiological characteristics in the resulting dopamine neurons while maintaining efficient cell production through established differentiation protocols

Inventive Principle:

Principle #10Preliminary action

2Adaptability or versatility

If cells with GBA1 variants are used for therapy, then patient-specific treatment is achieved, but GCase activity is reduced leading to disease progression

Engineering Contradiction:

Improvepatient-specific treatmentVSAvoidGCase activity

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent merges the patient-specific GBA1 variant allele with a wild-type GBA1 transgene in the pluripotent stem cells. This combination allows for patient-specific autologous cell therapy while simultaneously restoring GCase activity to normal or enhanced levels, preventing disease progression while maintaining personalized treatment benefits

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent converts the harmful effect of GBA1 variants into a benefit by using CRISPR-Cas9 to introduce a wild-type GBA1 transgene complementation approach. The patient-specific cells retain their genetic background and immunocompatibility advantages, while the introduced wild-type allele compensates for the pathogenic variant, transforming the disease-causing genetic background into a platform for effective therapy

Inventive Principle:

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

3Reliability

If GBA1 expression is increased using traditional methods, then GCase activity improves, but integration stability and consistency across cell populations deteriorate

Engineering Contradiction:

ImproveGCase activityVSAvoidtransgene integration stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent uses the Sleeping Beauty transposon system as an intermediary mechanism to integrate the GBA1 transgene. The transposase enzyme mediates stable integration of the transgene into the genome, ensuring consistent GCase activity across the cell population while maintaining genomic stability and preventing transgene loss during cell expansion and differentiation

Inventive Principle:

Principle #24Intermediary (Mediator)

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 results in increased GBA1 expression and GCase activity in stem cells, leading to the production of cells with improved physiological consistency and engraftment potential for therapeutic use in treating neurodegenerative diseases.

Implementation Method 1

introducing, into the cell, a transposase or a nucleic acid sequence encoding a transposase, wherein the introducing in (i) and (ii) results in integration of the DNA sequence encoding GBA1 into the genome of the cell

Methodology Applied

Scientific EffectTransposase-mediated integration: Enzyme

Data Source

PatentUS20230081881A1Transposon-based modulation of GBA1 and related compositions and uses thereof

Publication Date: 2023.03.16 ASPEN NEUROSCIENCE INC

AI summary

The present disclosure provides transposon-based methods of genetic editing in pluripotent stem cells, and methods of lineage specific differentiation of such edited pluripotent stem cells into floor plate midbrain progenitor cells, determined dopamine (DA) neuron progenitor cells, and/or DA neurons, or into glial cells, such as microglial cells, astrocytes, oligodendrocytes, or ependymocytes. Also provided are compositions and uses thereof, such as for treating neurodegenerative diseases and conditions, including Parkinson's disease.