HUMAN GENE THERAPY 11:1891–1900 (September 1, 2000) Mary Ann Liebert, Inc. Gene Electrotransfer Results in a High-Level Transduction of Rat Skeletal Muscle and Corrects Anemia of Renal Failure GABRIELLA RIZZUTO, MANUELA CAPPELLETTI, CARMELA MENNUNI, MACIEJ WIZNEROWICZ, ANNA DEMARTIS, DOMENICO MAIONE, GENNARO CILIBERTO, NICOLA LA MONICA, and ELENA FATTORI ABSTRACT We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroin- jection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secre- tion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be ap- plied to produce therapeutically significant levels of erythropoietin in chronic renal failure. 1891 OVERVIEW SUMMARY Anemia is a frequent complication of chronic renal failure; patients are currently successfully treated with recombinant EPO. Given the frequency of the treatments (three times a week) and the high cost of the protein, a gene therapy pro- tocol for the treatment of anemia in these patients would be of great advantage. We have previously demonstrated that a single intramuscular electroinjection of a plasmid DNA encoding the EPO cDNA in mice yields long-term produc- tion of significant amounts of EPO in circulation. Here we show that rat muscle can be electrically transduced with an efficiency comparable to that observed on treatment of the mouse tissue. In addition, we observed that a dose of 50 m g of plasmid DNA per kilogram of body weight can correct anemia induced by subtotal nephrectomy in rats. These re- sults further support the potential of gene electrotransfer for gene therapy applications. INTRODUCTION C HRONIC RENAL FAILURE (CRF) is characterized by a grad- ual and usually irreversible decline in kidney functions. In early stages of the disease CRF patients show several renal dys- functions, including anemia, but do not necessarily require reg- ular dialysis; in the late stages patients require regular dialysis or kidney transplantation for survival (Kerr, 1979). Therapy with human recombinant erythropoietin (EPO) has been accepted as effective for anemia in CRF patients (Esch- bach et al ., 1987). The current treatment consists of a starting dose of 50–100 U/kg three times a week; over this dose range 95% of all patients respond with a clinically significant increase in hematocrit (Eschbach et al ., 1989). When the hematocrit has reached the target value the maintenance dose is individually adjusted and is on average 75 units/kg three times weekly. In spite of the benefits of the treatment in correcting the hemato- logical and hemodynamic profile as well improving the pa- tient’s quality of life, the ability to deliver the hormone by a gene therapy approach would have substantial clinical and eco- nomic benefits. Several groups have shown the feasibility of using recombi- nant defective viral vectors, such as adenoviral (Svensson et al ., 1997), retroviral (Osborne et al., 1995), and adeno-associated virus (AAV)-based vectors (Descamps et al ., 1996; Kessler et al ., 1996; Snyder et al., 1997) to obtain sustained and prolonged production of EPO in the circulation; however, the limits im- posed on viral vectors by the host immune response are well documented (for review see Benihoud et al ., 1999). Previous infection with the wild-type virus can result in a neutralizing immune response, rendering ineffective the administration of the recombinant vector (Chirmule et al ., 1999; Erles et al ., Istituto di Richerche di Biologia Molecolare P. Angeletti, 00040 Pomezia, Rome, Italy.