1. ¹Institute of Hematology and Blood Transfusion, Prague, Czech Republic
2. ²Heinrich-Pette-Institute for Experimental Virology and Immunology at the University of Hamburg, Germany
3. ³Institute for Human Gene Therapy, Department of Molecular and Cellular Engineering, The Wistar Institute, University of Pennsylvania, Philadelphia, PA, USA
4. ⁴Department of Hematology and Oncology, Hannover Medical School, Hannover, Germany
5. ⁵Cincinnati Children's Hospital, Department of Experimental Hematology, Cincinnati, OH, USA
6. ⁶Baylor College of Medicine, Medicine – Hematology/Oncology, Houston, TX, USA

Correspondence: Dr J Jelinek, Department of Medicine-Hematology/Oncology, Baylor College of Medicine, One Baylor Plaza, Suite 802E, Houston, TX 77030, USA

The first two authors contributed equally

Received 3 July 2002; Accepted 23 November 2002.

Abstract

The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA. Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations. A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp. The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12. We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.