Strategies for Therapeutic Peptide Delivery by Mesenchymal Stem Cells

More than 50% of infants with ALL have leukemic blast cells that contain MLL fusion genes with reciprocal translocation t(4;11)(q21;q23). We have shown that AF4 and AF9 form a stable protein complex in the nucleus and that the mutual interaction domains of the two proteins are present within MLL fusion proteins (Erfurth et al., 2004). Mapping of the protein interaction domains reveals that a small, highly conserved portion of the AF4 molecule is necessary and sufficient to bind AF9. To test the significance of the AF4-AF9 protein interaction, we developed small synthetic peptides capable of interfering with AF4-AF9 binding in vitro. The peptides mimic the amino acid sequence of the AF9 binding domain within AF4 and compete with AF4 to block AF9 binding. We have shown that the synthetic peptide PFWT (P=Penetratin linked to FWT=LWVKIDLDLLSR) disrupts AF4-AF9 protein complexes and kills t(4;11) MV4;11 cells but not MOLT-4 leukemia cells or healthy CD34+ cells in vitro (Srinivasan et al., 2004). We are now developing new strategies for the use of MSC as an alternative to deliver recombinant FWT peptide directly to leukemia cells in an artificial local microrenvironment in vitro. In these new studies we designed HIV-1 based lentiviral vectors encoding FWT peptide (NL-FLAGHA2-FWT-9R) and CXCL12/FWT (NL-CXCL12-FCS-FLAG-HA2-FWT-9R) and examined whether recombinant peptide produced by modified MSC, modulate cell cycle and the growth kinetics of leukemic cells with t(4;11) translocation in vitro. Co-culture systems that promote direct cell-to-cell contact were first established with untransduced MSC or MSC modified to produce recombinant FWT and seeded with MV4;11 leukemia cells (1:1 ratio). MSC transduced with Lentiviral-Control vectors showed 2.8% killing of MV4;11 leukemia cells. The addition of suboptimal concentrations, 5μg/ml, synthetic PFWT peptide, caused 17.4% killing of MV4;11 leukemia cells. Recombinant FWT peptide produced endogenously by FWT-MSC also showed 19.5% killing. When suboptimal concentrations, 5 μg/ml synthetic PFWT peptide was added back to these co-cultures containing FWT-MSC, MV4;11 leukemia cell killing was further markedly enhanced >10 fold to 44% showing more than an addetive effect by synthetic PFWT peptide and endogenous recombinant FWT. Endogenous FWT produced by CXCL112/FWT MSC was also able to kill 20% of MV4;11 leukemia cells in the absence of PFWT synthetic peptide. Co-culture studies utilizing the control MOLT-4 leukemia cells that lack the t(4;11) translocation indicated that endogenous FWT produced by FWT transduced MSC or CXCL12/FWT transduced MSC, alone, or with additional 5μg/ml or 25μg/ml synthetic PFWT peptide, showed no significant killing of MOLT-4 leukemia cells. In summary, we have shown that FWT recombinant peptide is synthesized and released from MSC transduced with lentiviral vectors, NL-FLAG-HA2-FWT-9R or NL-CXCL12-FCS-FLAG-HA2-FWT-9R. The data demonstrate sufficient transfer of recombinant FWT peptide from both types of FWT-transduced MSCs to kill leukemic cells in vitro.These studies support the further development of MSC-mediated delivery of recombinant cytotoxic peptides to target leukemia.