Increased Gene Transfer into Human Cord Blood Cells by Centrifugation-Enhanced Transduction in Fibronectin Fragment-Coated Tubes

Abstract

We investigated whether transduction of human cord blood progenitor cells can be increased by spinoculation in fibronectin fragment CH-296 (FN)-coated tubes. Bicistronic vectors PA317/LgEIN, containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (neo) genes, and PG13/LgDIN, containing the dihydrofolate reductase and neo genes, were used to transduce CD34-enriched human cord blood cells. Transduction by spinoculation in FN-coated tubes (spin/FN⁺) was compared with spinoculation in noncoated tubes (spin/FN^-) and transduction in plates coated with FN(plate/FN⁺). Antibody to TGF- beta was added to spin/FN⁺ to evaluate its impact on transduction. Using producer cell line PA317/LgEIN for transduction of CD34⁺ cord blood cells, FACS analysis for expression of EGFP revealed mean transduction of 30.6 +/- 4.3, 9.1 +/- 1.6, and 21.1 +/- 6.5% of CD34⁺ cells in the spin/FN⁺, spin/FN^-, and plate/FN⁺ arms, respectively. Transduction of CD⁺CD38^low cells was also higher in the spin/FN⁺ arm as compared with transduction in the spin/FN^- arm. These results were corroborated by colony-forming assays. Antibody to TGF beta did not further increase transduction. Using a different producer cell line, PG13/pLgDIN, a higher number of G418-resistant CFU-GM was observed in the spin/FN + as compared with the plate/FN⁺ and spin/FN arms. NOD/SCID mice were transplanted with transduced, CD34-enriched human cord blood cells, and persistence of transduced human cells was analyzed in the mice marrows after 6-8 weeks: 32.8, 6.0, and 23.9% human G418-resistant CFU-GM colonies were observed in the spin/FN⁺, spin/FN^-, and plate/FN⁺ arms, respectively. These results suggest that spinoculation in FN-coated tubes increases transduction of early human cord blood progenitor cells as compared with spinoculation in noncoated tubes.