Ligand-regulatable erythropoietin production by plasmid injection and in vivo electroporation.
BACKGROUND
The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiency disorders. However, the ability to regulate transgene expression is of paramount importance for safe and effective gene transfer therapy.


METHODS
We developed two plasmids, one encoder of chimeric GeneSwitch protein, and the other an inducible transgene for human erythropoietin (Epo). The level of secretion of Epo into the serum was modulated by intraperitoneal administration of mifepristone (MFP). Rats were divided into four groups: one group administered Epo plasmid with MFP for 50 days, a second group administered Epo plasmid with MFP for 15 days and then again from day 30 to day 50, a third group administered Epo plasmid without MFP, and a fourth group administered control plasmid. A pair of electrodes was inserted into the muscle of the right thigh, 100 mg of each plasmid was injected, and in vivo electroporation (8 pulses at 100 V for 50 msec) was performed.


RESULTS
The presence of vector-derived Epo mRNA was detected by RT-PCR only in the Epo plasmid and MFP(+) groups. The hematocrit levels increased continuously, from the pre-injection level of 41.2% to 55.0% on day 30 and 53.8% on day 50 in the Epo plasmid and MFP(+) groups. In the MFP re-challenged group, the hematocrit levels rose up to day 15, fell after 20 to 30 days, and then rose again after MFP re-administration. The serum Epo levels increased only in the Epo plasmid and MFP(+) groups. There were no significant changes in hematocrit levels and Epo levels in the Epo plasmid and MFP(-) group.


CONCLUSIONS
Epo gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient drug-regulated delivery of Epo.
Terada
Yoshio
Y
Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, Tokyo, Japan. yterada.kid@tmd.ac.jp
Tanaka
Hiroyuki
H
Okado
Tomokazu
T
Shimamura
Haruko
H
Inoshita
Seiji
S
Kuwahara
Michio
M
Akiba
Takashi
T
Sasaki
Sei
S
D000818:Animals; D018274:Electroporation; D004921:Erythropoietin / Q000235:genetics* / Q000493:pharmacokinetics; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D006400:Hematocrit; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007273:Injections, Intramuscular; D018374:LLC-PK1 Cells; D008024:Ligands; D008297:Male; D008845:Microinjections; D015735:Mifepristone / Q000494:pharmacology; D010957:Plasmids / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013848:Thigh
D006727:Hormone Antagonists; D008024:Ligands; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D015735:Mifepristone