Pharmacological Activation of Guanine Nucleotide Exchange Factors for the Small GTPase Rap1 Recruits High-Affinity β1Integrins as Coreceptors for Parvovirus B19: ImprovedEx VivoGene Transfer to Human Erythroid Progenitor Cells

Abstract

Parvovirus B19 has potential as a gene therapy vector because of its restricted tropism for human erythroid progenitor cells in the bone marrow. B19 binds to the cell surface through P antigen and we identified activated β₁ integrins as coreceptors for internalization. Because differentiation with phorbol ester induces β₁ integrin coreceptor activity, but cell differentiation is not desirable in gene transfer to human progenitor cells and one of the downstream effectors of phorbol esters is the small GTPase Rap1, the role of Rap1 in the recruitment of β₁ integrins on hematopoietic cells was examined. Expression of a constitutively active Rap1 (63E) was sufficient to recruit β₁ integrin coreceptors in erythroleukemic K562 cells by inducing high-affinity integrin conformation. A crucial role of actin polymerization in Rap1-mediated β₁ integrin recruitment was documented by complete inhibition of the 63E Rap1 effect with low-dose cytochalasin D and by the ability of a constitutively active mutant of the actin cytoskeleton regulator Rac1 to sensitize K562 cells to the pharmacological activation of endogenous Rap1, using the Rap1 exchange factor-specific 8-pCPT-2′-O-Me-cAMP [8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate]. Interestingly, in primary human erythroid progenitor cells, 8-pCPT-2′-O-Me-cAMP was sufficient to significantly increase B19-mediated gene transfer, suggesting that these cells possess the cytoskeleton organization capacity required for efficient recruitment of β₁ integrins by brief pharmacological stimulation of Rap1 GTP loading. Because 8-pCPT-2′-O-Me-cAMP has been implicated in enhanced homing of progenitor cells, these results identify a novel tool with which to optimize ex vivo B19-mediated gene transfer and potentially improve homing of transduced cells by Rap1–β₁ integrin activation with 8-pCPT-2′-O-Me-cAMP.