Transduction of Human CD34+ Cells with Recombinant TAT-NF-YA Protein Enhances Their Ex Vivo Expansion and Repopulating Potential in NOD/SCID Mice

Protein transduction offers great promise for clinical applications as a safe alternative to viral gene delivery. We have previously shown that NF-YA transcription factor which regulates the expression of several genes implicated in hematopoietic stem cell (HSC) renewal and differentiation, including the Hox4 paralogs HoxB4, HoxC4, HoxD4, LEF1, Notch1, p27 and telomerase (

1. stimulates the ex vivo expansion of hematopoietic precursor and progenitor cells and

2. maintains and/or expands the repopulating capacity of HSC in the NOD/SCID mouse model.

Human CD34+ cells from peripheral blood or bone marrow were treated with IL3/IL6/SCF/FL/TPO, with or without TAT-NF-YA protein, and grown for up to two weeks in a serum free media. Fifteen days after fusion protein delivery cell growth in treated culture was 4-fold higher than in control cultures without added TAT-NF-YA. Cells plated on methylcellulose at different time points of cell growth showed an increase of colony-forming progenitor cells (CFC) in culture exposed to TAT-NF-YA primarily due to increase of GEMM population: ~3 fold at day 4 and 7-fold day 9 after protein delivery. These results suggest that TAT-NF-YA promotes cell proliferation by maintaining the most primitive population in an undifferentiated state, biasing HSC toward self-renewal rather than differentiation. To measure more directly the engraftment potential of TAT-NF-YA treated cells, bone marrow CD34+ cells cultured for 3 or 6 days after exposure to the protein were transplanted into NOD/SCID mice. After 9 weeks, human CD45+ donor cells were analyzed for myeloid and lymphoid surface markers in the bone marrow of recipient mice by flow cytometry with anti-human CD33 and CD19 antibodies. TAT-NF-YA plus cytokine treated cells demonstrated significantly higher long-term repopulating capacity of cultured cells compared to cells cultured with cytokines alone, averaging a 4-fold increase for 3-day and a 3-fold increase for 6-day cell culture in the percent of huCD45+ cells recovered from the circulation and spleen of NOD/SCID mice. The proportion of lymphoid and myeloid engrafted populations was not significantly different in control and protein treated cells. These data provide evidence that NF-YA delivered as a TAT fusion protein to primary hematopoietic cells may stimulate progenitor/stem cell expansion, homing and/or repopulating ability. More generally, these results indicate that protein transduction has significant potential for various applications in manipulation of HSC, thereby offering a potentially safer alternative to viral gene therapy; both because the protein has a shot half-life in the targeted cells in vivo and because there is opportunity for insertional mutagenesis.