Methods of making red blood cells and platelets in vitro and uses thereof
Inventors
Assignees
University of Utah • University of Utah Research Foundation Inc
Publication Number
US-12139724-B2
Publication Date
2024-11-12
Expiration Date
2036-07-13
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Abstract
Disclosed herein are methods of producing platelets and red blood cells using synthetic biology and uses thereof. The methods disclosed herein can also be used to produce platelets and red blood cells comprising a therapeutic agent. The cells produced by the methods disclosed herein can be used to treat, manage, prevent and diagnosis various diseases and disorders and be used as a research tool.
Core Innovation
The invention provides methods for producing platelets and red blood cells in vitro using synthetic biology techniques. Specifically, these methods utilize genetically engineered feeder and fed cells, each containing distinct genetic circuits with specific genes of interest and promoters. By culturing the genetically engineered feeder cells together with the genetically engineered fed cells under suitable conditions, the fed cells are induced to differentiate into red blood cells or platelets, enabling controlled cell production.
These methods can also result in the production of platelets and red blood cells that comprise therapeutic agents encoded by inserted genes within the genetic circuits. The design enables modulation of cell differentiation through both extrinsic (environmental) and intrinsic (transcription factor-based) control, leveraging genetic switches, recombinases, and regulatory elements integrated into the cells. Precise temporal and spatial gene expression control is achieved via media modulators and inducible promoters.
The background outlines the challenges in current approaches, including the difficulties in expanding and controlling hematopoietic stem cells (HSCs) in vitro, insufficient production capacity from alternative cell sources, and the need to better mimic endogenous bone marrow environments. The invention addresses these issues by developing robust, modular, and scalable systems for the efficient, controlled production of functional platelets and red blood cells, which may also include therapeutic functionalities.
Claims Coverage
The patent claims one main inventive feature for the method of producing platelets or red blood cells comprising a therapeutic peptide via engineered genetic circuits and culture techniques.
Method for producing platelets or red blood cells comprising a therapeutic peptide using genetically engineered fed cells with specific genetic circuits
The method involves: 1. Providing a feeder cell. 2. Providing a genetically engineered fed cell that contains: - A first genetic circuit with GATA-1 and/or HoxB4, one or more recombination sites, one or more recombinases, and one or more promoters operably linked to these components. - A second genetic circuit comprising a gene of interest capable of encoding a therapeutic peptide, also with one or more recombination sites, recombinases, and promoters operably linked to the gene of interest. 3. Culturing the feeder cell with the genetically engineered fed cell under conditions permitting differentiation into platelet progenitor stem cells and/or red blood progenitor stem cells with expression of the gene of interest. 4. Producing platelet progenitor stem cells and/or red blood progenitor stem cells comprising the therapeutic peptide encoded by the gene of interest. The method optionally includes: - Re-culturing the progenitor cells to induce their differentiation into platelets and/or red blood cells. - Collecting and isolating the differentiated platelets and red blood cells. Key features include: - Regulation of the genetic circuits via media modulators (such as IPTG, tetracycline, doxycycline, quinic acid, or auxin). - Use of specific promoters (CMV, RSV, and/or U6). - Employment of recombinases (Cre or phiC31 integrase) and recombination sites (loxP, attP, Bxb1), which may be positioned at the Rosa26 locus. - Repressor proteins (LacI, TetR, QS) may also be included in the circuits. - The method applies to fed cells derived from embryonic stem cells, mouse embryonic stem cells, or hematopoietic progenitor stem cells from various sources.
The inventive features are centered on the use of specified genetic circuits within engineered cells to direct the in vitro differentiation and production of platelets or red blood cells carrying therapeutic peptides, with tightly regulated gene expression and differentiation controlled by synthetic biology tools.
Stated Advantages
The methods enable large-scale and efficient in vitro production of functional, purified platelets and red blood cells, potentially from autologous sources.
In vitro production reduces risks associated with human donor dependence and transmission of donor-related complications.
The system provides precise temporal and spatial regulation of gene expression, improving control over cell differentiation processes.
Platelets and red blood cells can be engineered to include or deliver therapeutic agents, enhancing their functionality for therapeutic applications.
Synthetic biology circuits allow for modular, robust, and predictable manipulation of cell fate and therapeutic payload delivery.
Documented Applications
Treatment, management, prevention, and diagnosis of various diseases and disorders using in vitro produced red blood cells or platelets.
Use of engineered platelets and red blood cells as a research tool for studying cell fate decisions, differentiation, and the bone marrow environment.
Therapeutic administration for patients needing platelet or red blood cell transfusions.
Use of engineered platelets to deliver therapeutic agents, such as enzymes to break down plaque in coronary arteries or to act as diagnostic indicators by secreting detectable reporters.
Application of engineered cells to deliver cytokines or angiostatins to target sites, such as in cancer therapy.
Use of synthetic biology-engineered platelets and red blood cells for in vivo delivery of therapeutic or diagnostic biomolecules.
Probing neurological disorders and aiding the diagnosis and study of neurodegenerative diseases by reprogramming cells with genetic circuits.
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