Careful adjustment of Epo non-viral gene therapy for β-thalassemic anaemia treatment
© Fabre et al; licensee BioMed Central Ltd. 2008
Received: 12 September 2007
Accepted: 11 March 2008
Published: 11 March 2008
In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in β-thalassemic mice.
Muscular electrotransfer of 1 μg, 1.5 μg, 2 μg 4 μg or 6 μg of Epo-plasmid was performed in β-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution.
Muscular electrotransfer of the 1.5 μg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in β-thalassemic mice for more than 9 months.
This strategy led to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments.
Therapeutic protein secretion by an in vivo transfected organ is one of the major gene therapy applications. One drawback to be avoided in such therapeutic strategy is the potentially deleterious secretion peak of therapeutic protein following DNA administration. The aim of this study was to adapt dosage and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak.
Recombinant erythropoietin (rhEpo) injections are commonly used to treat anaemia linked to cancer treatment or chronic renal failure. However, rhEpo injections remain an expensive treatment which requires frequent delivery injection repeats and which can lead to anti-Epo antibodies production by the patient . Therefore, erythropoietin (Epo) gene transfer appears to be a promising alternative for severe anaemia treatment since it requires less frequent treatment repeat and may allow sustained Epo secretion and constant patient coverage. Epo gene transfer has already been tested on normal animals and on anaemia animal models such as β-thalassemia and chronic renal failure models. To this end, various gene transfer strategies have been used such as ex-vivo strategies using engrafted transduced myoblasts or other cell types [2–4], viral strategies using adenovirus  adeno-associated virus [6, 7], helper-dependent adenovirus , or non-viral strategies using naked DNA injection , poloxamer/DNA formulations  or naked DNA injection associated to electrotransfer [9, 11–13]. In several of these studies, the gene dose transferred led to a maximum hematocrit value between 70 and 80% [6, 9–13] which corresponds to potentially lethal polycythemia . Therefore, in the particular case of Epo, an accurate level of transgene expression is required for safety reasons.
Temporal control systems of transgene expression have already been used in gene therapy preclinical experiments, including for Epo gene use [6, 10, 14, 15]. These systems could avoid deleterious Epo secretion peak, but unsolved problems such as host immune response against the transactivator  or inducing agents adverse effects, are still restricting their use.
In order to avoid the toxic Epo plasmatic peak and to reduce plasmatic fluctuation amplitude, we decided to test different doses and administration schedules of an Epo encoding plasmid in anaemia treatment of β-thalassemic mice. Considering electrotransfer advantages in terms of safety, efficiency and cost, we chose this well-handled gene transfer method. Our previous experiment with β-thalassemic mice using intramuscular electrotransfer of an Epo encoding plasmid  led to a first estimation of transgene product kinetics and physiologic effects. Epo plasmatic level was found to reach a peak value within two weeks after gene therapy treatment and then to decrease approximately of 40%, 20% and 15% of this peak after 1, 2 and 3 months, respectively. This plasmatic Epo kinetics was roughly confirmed in normal mice by other studies with a secretion peak one week after electrotransfer [11, 13]. However, Epo main physiologic effect on erythropoiesis which can be evaluated through hematocrit measurement remained intense for several months because of red blood cell half-life. Indeed, β-thalassemic mice hematocrit was still at the polycythemic value of 60% four months after 20 μg Epo-plasmid electrotransfer .
Considering those results, we have presently tested the therapeutic potential of repeated electrotransfer of suboptimal low Epo-plasmid doses in the β-thalassemic mouse model to restore and maintain a normal hematocrit without reaching toxicity.
The pCMV-Epo plasmid used for experiments was a pCOR plasmid  containing the mouse erythropoietin cDNA under the regulatory control of the hCMV E/P . Plasmid large-scale production and double caesium chloride gradient ultracentrifugation used as purification method, were realised according to traditional molecular biology methods . Plasmid construct was checked by restriction fragment length profile and sequencing.
Animal experiments were conducted following NIH recommendations. The β-thalassemic Hbb-thal1 mice  from the laboratory of Haematopoietic Gene Therapy (Saint Louis Hospital, Paris, France) were used for experiments. Two to four months female mice were separated into 6 groups: six Hbb-thal1 mice per group were used for the higher plasmid dose experiment, and eight Hbb-thal1 mice per group were used for the lower plasmid dose experiment. Mice were first anaesthetised by intra-peritoneal injection of 250 μl of a ketamine-xylazine solution (respectively 8.66 mg/ml and 0.31 mg/ml in 150 mM NaCl). Left rear legs were shaved and the Epo-plasmid solution was injected in the tibialis-cranialis muscle. The DNA solutions were diluted in 150 mM NaCl to contain the desired plasmid quantity in 30 μl: 1 μg, 1.5 μg, 2 μg, 4 μg and 6 μg, respectively, for the corresponding groups (meaning 50, 75, 100, 200 or 300 ng of plasmid per mouse gram, respectively). The DNA injection was immediately followed by application of eight electric pulses of 200 V/cm intensity, 20 ms duration and delivered at a frequency of 1 Hz, using plate electrodes and generator BTX ECM 830 (Genetronics™), as previously described .
Sample collection, measurement and assay
Blood samples were collected by retro-orbital puncture of anaesthetised mice at desired time after plasmid electrotransfer. Hematocrits were measured using a standard micro-hematocrit method . Mouse Epo assay was realised on serum samples using the EPO ELISA Medac® kit (Medac™) based on cross-reaction with human Epo.
Analysis of variance (ANOVA) and Fisher PLSD were used.
Results and discussion
Our previous study of β-thalassemic mice demonstrated that electrotransfer of 1–10 μg Epo-plasmid doses were sufficient to induce a significant hematocrit increase. However, after a hematocrit burst depending on the dose of injected DNA during the first month after treatment, the hematocrit of treated mice started to decrease, and finally stabilised two months after electrotransfer. Surprisingly, this plateau was the same whatever the DNA dose used for gene transfer, and hematocrit still remained different from controls for at least 4 months . Moreover, the 5 μg Epo-plasmid dose seemed to be the most appropriate since it led to normal hematocrit at peak value (approximately 45%). This hematocrit profile resulted from a shorter Epo plasmatic kinetics with peak of expression reached in less than 2 weeks and an expression level relative to this peak value of 40%, 20% and 15% respectively 1, 2 and 3 months after electrotransfer. Higher doses of Epo-plasmid led to hazardous unsafe hematocrit peak (60 to 80%). This study is then designed to slowly reach and maintain the hematocrit plateau and to avoid the initial hemarocrit burst.
This over one year study indicates that an appropriate administration schedule to treat β-thalassemic anaemia in mice could consist in a 1.5 μg Epo-plasmid dose electrotransfer firstly repeated after 5 weeks as an initiating dose to restore a normal hematocrit, and then repeated every 3 or 4 months to maintain this hematocrit level. The present experiment shows that repeated electrotransfer of low Epo-plasmid doses allows fine tuning of hematocrit response on a more than one year period. Looking at individual data, it appears that the hematocrit can be maintained at an almost constant level for each of the treated animal. This strategy allows to avoid the deleterious initial hematocrit peak and to maintain a normal hematocrit with small fluctuation amplitude. Furthermore, we may hypothesise that this administration schedule which leads to low Epo endogenous production, may limit humoral response which has been clearly correlated to transgene expression level . Therefore, anti-Epo antibodies production coming along with host autoimmune reaction, which has already been described in non-human primate , might be avoided with the present repeated and light therapeutic protocol.
Regarding possible clinical applications of the electrotransfer technology, one may argue that repetitive use of electric pulses might be painful. As far as we know, no significant discomfort related to the electrotransfer technology in humans has been reported so far. Several clinical trials of electrochemotherapy were reported with a good tolerance to the electric pulses delivery. Electrochemotherapy has recently been evaluated in an European project (ESOPE) and validated for clinical use.
As far as muscle electrotransfer is concerned, at least two clinical trials have been approved and are being conducted in the area of cancer vaccination by two different companies, Ichor and Inovio (vaccination using tumor antigen). The results of these first in man studies should give us more details about the discomfort linked to this procedure.
The present work indicates that plasmids can be delivered repetitively with little or none impairment of transgene delivery and expression, in opposite to viral vector mediated gene delivery. This repeated delivery protocol allows careful adjustments to reach the clinical endpoint and feedback for subsequent dose delivery. This safe treatment protocol could be applied to another anaemic context and extend to a wide variety of gene therapy applications using many candidate therapeutic genes such as growth factor genes.
The authors thank Michael Bettan for preliminary study of β-thalassemic mice treatment with Epo-plasmid muscular electrotransfer. The authors acknowledge the Association Française contre les Myopathies (AFM) for its financial support.
- Macdougall IC: Antibody-mediated pure red cell aplasia (PRCA): epidemiology, immunogenicity and risks. Nephrol Dial Transplant. 2005, 20 Suppl 4: iv9-15. 10.1093/ndt/gfh1087.PubMedGoogle Scholar
- Sommer B, Rinsch C, Payen E, Dalle B, Schneider B, Deglon N, Henri A, Beuzard Y, Aebischer P: Long-term doxycycline-regulated secretion of erythropoietin by encapsulated myoblasts. Mol Ther. 2002, 6 (2): 155-161. 10.1006/mthe.2002.0646.View ArticlePubMedGoogle Scholar
- Orive G, De Castro M, Ponce S, Hernandez RM, Gascon AR, Bosch M, Alberch J, Pedraz JL: Long-term expression of erythropoietin from myoblasts immobilized in biocompatible and neovascularized microcapsules. Mol Ther. 2005, 12 (2): 283-289. 10.1016/j.ymthe.2005.04.002.View ArticlePubMedGoogle Scholar
- Lippin Y, Dranitzki-Elhalel M, Brill-Almon E, Mei-Zahav C, Mizrachi S, Liberman Y, Iaina A, Kaplan E, Podjarny E, Zeira E, Harati M, Casadevall N, Shani N, Galun E: Human erythropoietin gene therapy for patients with chronic renal failure. Blood. 2005, 106 (7): 2280-2286. 10.1182/blood-2004-11-4174.View ArticlePubMedGoogle Scholar
- Osada S, Ebihara I, Setoguchi Y, Takahashi H, Tomino Y, Koide H: Gene therapy for renal anemia in mice with polycystic kidney using an adenovirus vector encoding the human erythropoietin gene. Kidney Int. 1999, 55 (4): 1234-1240. 10.1046/j.1523-1755.1999.00381.x.View ArticlePubMedGoogle Scholar
- Johnston J, Tazelaar J, Rivera VM, Clackson T, Gao GP, Wilson JM: Regulated expression of erythropoietin from an AAV vector safely improves the anemia of beta-thalassemia in a mouse model. Mol Ther. 2003, 7 (4): 493-497. 10.1016/S1525-0016(03)00043-1.View ArticlePubMedGoogle Scholar
- Chenuaud P, Larcher T, Rabinowitz JE, Provost N, Cherel Y, Casadevall N, Samulski RJ, Moullier P: Autoimmune anemia in macaques following erythropoietin gene therapy. Blood. 2004, 103 (9): 3303-3304. 10.1182/blood-2003-11-3845.View ArticlePubMedGoogle Scholar
- Maione D, Wiznerowicz M, Delmastro P, Cortese R, Ciliberto G, La Monica N, Savino R: Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector. Hum Gene Ther. 2000, 11 (6): 859-868. 10.1089/10430340050015473.View ArticlePubMedGoogle Scholar
- Payen E, Bettan M, Rouyer-Fessard P, Beuzard Y, Scherman D: Improvement of mouse beta-thalassemia by electrotransfer of erythropoietin cDNA. Exp Hematol. 2001, 29 (3): 295-300. 10.1016/S0301-472X(00)00679-2.View ArticlePubMedGoogle Scholar
- Richard P, Pollard H, Lanctin C, Bello-Roufai M, Desigaux L, Escande D, Pitard B: Inducible production of erythropoietin using intramuscular injection of block copolymer/DNA formulation. J Gene Med. 2005, 7 (1): 80-86. 10.1002/jgm.631.View ArticlePubMedGoogle Scholar
- Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P, Mathiesen I, Cortese R, Ciliberto G, Laufer R, La Monica N, Fattori E: Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci U S A. 1999, 96 (11): 6417-6422. 10.1073/pnas.96.11.6417.PubMed CentralView ArticlePubMedGoogle Scholar
- Maruyama H, Ataka K, Gejyo F, Higuchi N, Ito Y, Hirahara H, Imazeki I, Hirata M, Ichikawa F, Neichi T, Kikuchi H, Sugawa M, Miyazaki J: Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats. Gene Ther. 2001, 8 (6): 461-468. 10.1038/sj.gt.3301412.View ArticlePubMedGoogle Scholar
- Fattori E, Cappelletti M, Zampaglione I, Mennuni C, Calvaruso F, Arcuri M, Rizzuto G, Costa P, Perretta G, Ciliberto G, La Monica N: Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates. J Gene Med. 2005, 7 (2): 228-236. 10.1002/jgm.652.View ArticlePubMedGoogle Scholar
- Lamartina S, Roscilli G, Rinaudo CD, Sporeno E, Silvi L, Hillen W, Bujard H, Cortese R, Ciliberto G, Toniatti C: Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators. Hum Gene Ther. 2002, 13 (2): 199-210. 10.1089/10430340252769734.View ArticlePubMedGoogle Scholar
- Trollet C, Ibanez-Ruiz M, Bloquel C, Valin G, Scherman D, Bigey P: Regulation of Gene Expression Using a Conditionnal RNA Antisense Strategy. J Genome Sci Tech. 2004, 3: 1-13. 10.1166/gl.2004.036.View ArticleGoogle Scholar
- Soubrier F, Cameron B, Manse B, Somarriba S, Dubertret C, Jaslin G, Jung G, Caer CL, Dang D, Mouvault JM, Scherman D, Mayaux JF, Crouzet J: pCOR: a new design of plasmid vectors for nonviral gene therapy. Gene Ther. 1999, 6 (8): 1482-1488. 10.1038/sj.gt.3300968.View ArticlePubMedGoogle Scholar
- Kreiss P, Bettan M, Crouzet J, Scherman D: Erythropoietin secretion and physiological effect in mouse after intramuscular plasmid DNA electrotransfer. J Gene Med. 1999, 1 (4): 245-250. 10.1002/(SICI)1521-2254(199907/08)1:4<245::AID-JGM49>3.0.CO;2-G.View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. Edited by: Press CSHL. 1989, New YorkGoogle Scholar
- Skow LC, Burkhart BA, Johnson FM, Popp RA, Popp DM, Goldberg SZ, Anderson WF, Barnett LB, Lewis SE: A mouse model for beta-thalassemia. Cell. 1983, 34 (3): 1043-1052. 10.1016/0092-8674(83)90562-7.View ArticlePubMedGoogle Scholar
- Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D: High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci U S A. 1999, 96 (8): 4262-4267. 10.1073/pnas.96.8.4262.PubMed CentralView ArticlePubMedGoogle Scholar
- Koepke JA: Practical Laboratory Hematology. 1991, New York , Churchill LivingstoneGoogle Scholar
- Popp RA, Popp DM, Johnson FM, Skow LC, Lewis SE: Hematology of a murine beta-thalassemia: a longitudinal study. Ann N Y Acad Sci. 1985, 445: 432-444. 10.1111/j.1749-6632.1985.tb17213.x.View ArticlePubMedGoogle Scholar
- de Franceschi L, Rouyer-Fessard P, Alper SL, Jouault H, Brugnara C, Beuzard Y: Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy. Blood. 1996, 87 (3): 1188-1195.PubMedGoogle Scholar
- Brugnara C: Iron deficiency and erythropoiesis: new diagnostic approaches. Clin Chem. 2003, 49 (10): 1573-1578. 10.1373/49.10.1573.View ArticlePubMedGoogle Scholar
- Beguin Y, Loo M, R'Zik S, Sautois B, Lejeune F, Rorive G, Fillet G: Effect of recombinant human erythropoietin on platelets in patients with anemia of renal failure: correlation of platelet count with erythropoietic activity and iron parameters. Eur J Haematol. 1994, 53 (5): 265-270.View ArticlePubMedGoogle Scholar
- Lee AH, Suh YS, Sung JH, Yang SH, Sung YC: Comparison of various expression plasmids for the induction of immune response by DNA immunization. Mol Cells. 1997, 7 (4): 495-501.PubMedGoogle Scholar
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