Transduction of Human CD34+ Cells with Recombinant TAT-NF-YA Protein Enhances Their Ex Vivo Expansion and Repopulating Potential in NOD/SCID Mice
Authors: Domashenko Alevtina , Danet-Desnoyers Gwenn , Sundby Russell , Emerson Stephen G. 
Published: October 2019

Abstract
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 (Zhu J. et al. PNAS, 102:11728–11733; 2005) can be delivered as a TAT-NF-YA fusion protein to primitive human hematopoietic CD34+ cells (Domashenko A. et al. Blood, 106:144B; 2005). In these earlier studies transduced human NF-YA protein demonstrated functional activity by upregulated HOXB4 mRNA, as measured by real time PCR. Based on these results we have now asked whether TAT-NF-YA treatment of CD34+ cells:
stimulates the ex vivo expansion of hematopoietic precursor and progenitor cells and 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.