Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development

Area of Science:

• Gene therapy
• Retroviral vectors
• Stem cell biology

Background:

• Murine stem cell virus (MSCV) vectors efficiently express transgenes in undifferentiated embryonic stem cells (ESCs).
• However, transgene expression dramatically decreases during in vitro hematopoietic differentiation of ESCs.
• This limits the utility of MSCV vectors for applications requiring sustained expression in differentiated hematopoietic cells.

Purpose of the Study:

• To construct and characterize a self-inactivating gammaretroviral vector derived from MSCV.
• To improve sustained transgene expression in ESCs and their differentiated progeny, particularly hematopoietic cells.
• To evaluate the vector's performance in both in vitro differentiation models and in vivo transplantation settings.

Main Methods:

• Construction of a self-inactivating MSCV-derived vector, MSinSB, featuring an embedded intron within the transgene cassette.
• Utilizing a composite cytomegalovirus immediate early enhancer-chicken beta-actin promoter for the internal transgene unit.
• Production of MSinSB at high titers and assessment of transgene expression in ESCs, differentiated hematopoietic cells, and murine bone marrow transplant recipients.

Main Results:

• MSinSB achieved high titers (approaching 10^6 transducing units/ml) and superior transgene expression in ESCs compared to a splicing-optimized vector (MSGV1).
• MSinSB demonstrated sustained transcriptional activity in >90% of differentiated hematopoietic cells, whereas MSGV1 expression was nearly abolished.
• Persistent high-level transgene expression was confirmed in vivo in murine bone marrow transplant recipients and in human CD34(+) cord blood stem/progenitor cells undergoing myelomonocytic differentiation.

Conclusions:

• The self-inactivating MSinSB vector effectively overcomes the silencing of transgene expression observed with conventional MSCV vectors during hematopoietic differentiation.
• MSinSB provides a promising tool for achieving persistent high-level transgene expression in stem cells and their differentiated derivatives.
• This vector holds potential for gene therapy applications requiring long-term expression in hematopoietic lineages.