Vectors and methods for the efficient generation of integration/transgene-free induced pluripotent stem cells from peripheral blood cells
Inventors
Baylink, David J. • Lau, Kin-Hing William • Zhang, Xiaobing
Assignees
Publication Number
US-9850499-B2
Publication Date
2017-12-26
Expiration Date
2033-05-21
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Abstract
A vector for generating induced pluripotent stem cells from human target cells comprising a) a vector backbone, b) exactly two, three or four transcription and reprogramming factor genes, each gene separated by a 2a self-cleavage peptide sequence, c) a spleen focus-forming virus promoter, and d) a post-transcriptional regulatory element Wpre, with or without an anti-apoptotic factor gene. A method for generating integration-free induced pluripotent stem cells, the method comprising: a) providing target cells, b) providing one or more than one vector according to the present invention, c) transducing or transfecting the target cells with the one or more than one vector, and d) culturing the transduced or transfected cells in a cell culture, thereby generating integration-free induced pluripotent stem cells.
Core Innovation
The invention provides vectors and methods for generating integration/transgene-free induced pluripotent stem cells (iPSCs) from human target cells such as peripheral blood cells and hematopoietic stem cells. The core vector includes a vector backbone, exactly two, three, or four transcription and reprogramming factor genes (for example, oct4 and sox2, optionally with klf4 and myc), each separated by a 2a self-cleavage peptide sequence, a spleen focus-forming virus (SFFV) promoter, and a post-transcriptional regulatory element Wpre. The vector may optionally include an anti-apoptotic factor gene, such as bcl-xl or bcl2.
The problem addressed is the inefficiency, complexity, and safety concerns associated with prior methodologies for generating integration/transgene-free iPSCs. Previous approaches relied on viral-based integration methods or required use of multiple reprogramming factors, some of which are oncogenic or otherwise raise clinical safety concerns, and produced low colony yields or involved labor-intensive procedures such as daily mRNA addition, virus production, or additional transgene excision steps. Additionally, T and B cell-derived iPSCs have genetic rearrangements that limit their usefulness, and existing methods from non-lymphoid blood cells achieved ineffcient reprogramming.
The disclosed vectors and methods allow efficient generation of integration/transgene-free iPSCs from enriched or depleted populations of peripheral blood cells without the use of excessive or potentially unsafe reprogramming factors. The approach uses episomal vectors or other non-integrating backbones and exploits strong promoters, regulatory elements, and anti-apoptotic factors to increase reprogramming efficiency to levels suitable for clinical or therapeutic applications. The method optionally includes steps for enriching the cell population for optimal reprogramming and culturing conditions to further enhance colony formation.
Claims Coverage
The patent has two independent claims outlining the inventive features for vectors and methods in the field of induced pluripotent stem cell generation.
Vector with specific reprogramming factors and regulatory elements
A vector for generating induced pluripotent stem cells from human target cells incorporates: - A vector backbone - At least two transcription and reprogramming factor genes, including oct4 and sox2, separated by a 2a self-cleavage peptide sequence - A spleen focus-forming virus (SFFV) promoter - A post-transcriptional regulatory element Wpre This core design may optionally include: - The vector backbone as an oriP/EBNA1-based episomal vector or plasmid backbone - The at least two transcription and reprogramming factor genes being exactly three (oct4, sox2, klf4) or exactly four (oct4, sox2, klf4, myc) - Use of a 2a self-cleavage peptide from a specified virus - Addition of anti-apoptotic factor genes such as BCL-XL or BCL2.
Method for generating integration-free induced pluripotent stem cells using the vector
A method for generating integration-free induced pluripotent stem cells comprising the steps: 1. Providing hematopoietic target cells 2. Providing one or more than one vector as described above 3. Transducing or transfecting the hematopoietic target cells with the vector(s) 4. Culturing the transduced or transfected cells in a cell culture to generate integration-free induced pluripotent stem cells This method may further comprise: - Using one or multiple vectors in differing ratios - Including additional vectors with specified reprogramming or anti-apoptotic genes - Harvesting target cells from body fluids such as peripheral blood - Enriching or purifying for specific cell markers (e.g., CD33) - Culturing cells for 3 to 6 days, particularly 4 days, prior to transduction or transfection.
In summary, the inventive features focus on a vector with a defined set of reprogramming genes and regulatory elements for efficient, integration-free iPSC generation and a corresponding method applying these vectors to hematopoietic cells using optimized cell preparation and culturing steps.
Stated Advantages
Efficient production of integration/transgene-free induced pluripotent stem cells from peripheral blood or hematopoietic cells.
Requires fewer reprogramming factors than prior methods, avoiding the need for excessive or oncogenic reprogramming genes.
Does not require transgene excision or daily mRNA application, making the process less labor-intensive and more convenient.
Yields colony numbers suitable for therapeutic applications (20–30 colonies/ml peripheral blood), which is up to 1000-fold higher than previous methods.
The iPSCs generated are morphologically, phenotypically, and functionally normal and can differentiate into multiple cell types.
Applicable to allogeneic cell therapy, disease modeling, and iPSC banking due to the integration-free nature and origin from non-lymphoid cells.
Documented Applications
Generation of patient-specific induced pluripotent stem cells for use in regenerative medicine.
Allogeneic cell therapy using integration-free iPSCs for therapeutic interventions.
Disease modeling with iPSCs derived from non-lymphoid blood cells.
Induced pluripotent stem cell banking for future clinical or research use.
Treatment of conditions or diseases in humans, including autoimmune diseases, cancer, cardiovascular disease, connective tissue diseases, injuries, and neurodegenerative diseases using integration-free iPSCs.
Differentiation of iPSCs generated by the disclosed method into mature cell types such as cardiomyocytes, hepatocytes, mesenchymal stem cells, and neurons.
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