RSS-Feed abonnieren
DOI: 10.1055/s-2002-25151
Chances and Shortcomings of Adenovirus-Mediated ATP7B Gene Transfer in Wilson Disease: Proof of Principle Demonstrated in a Pilot Study with LEC rats
Möglichkeiten und Grenzen von adenoviralem Gentransfer bei Morbus Wilson: Demonstration des Therapieprinzips in einer Pilotstudie an LEC-RattenPublikationsverlauf
11.9.2001
23.11.2001
Publikationsdatum:
17. April 2002 (online)

Abstract
Background & Aims: Wilson disease (WD) is an autosomal recessively inherited copper storage disease due to mutations in the ATP7B gene. It results in impaired biliary copper excretion followed by liver injury leading to cirrhosis. In parallel, copper accumulates in other tissues e. g. basal ganglia of the brain inducing motoric disorders. Phenotypical cure of Wilson disease by liver transplantation raised the question whether gene therapy may represent a successful alternative treatment procedure. To examine the principle feasibility of this approach we investigated the effects of gene transfer using an adenoviral vector construct expressing the human ATP7B cDNA in an established rodent model for WD, the Long-Evans Cinnemon rat (LEC).
Methods: Transduction efficiency was assessed by RT-PCR, Western blot and immunofluorescence analysis. The therapeutic effect was estimated by analyzing holoceruloplasmin and its ferroxidase activity in serum, and the copper content of excrements. Changes in copper homeostasis were determined by positron emission tomography (PET).
Results: Successful, but temporary gene transfer was clearly detectable on RNA and protein levels. In parallel the temporary therapeutic effect was documented by restoration of serum holoceruloplasmin and of its ferroxidase activity. Additionally the Ad-ATP7B treated LEC rat revealed a higher 64Cu content in stool. PET was able to visualize differences in 64Cu distribution between wild type and LEC rats, indicating its principle usefulness as analytical tool.
Conclusion: The data demonstrate proof of principle of successful gene therapy in an experimental model of WD. As a consequence of successful but only transient therapeutic effect of adenoviral gene transfer we can now focus more efficient and permanent gene transfer strategies.
Zusammenfassung
Hintergrund und Ziel: Der autosomal-rezessiv vererbte Morbus Wilson (WD) ist eine Kupferspeichererkrankung aufgrund von Mutationen im ATP7B-Gen. Er ist gekennzeichnet durch eine verminderte biliäre Kupferausscheidung mit nachfolgender Leberschädigung bis hin zur Zirrhose sowie einer Akkumulation von Kupfer auch in anderen Geweben. Aufgrund der Tatsache, dass der Morbus Wilson durch eine Lebertransplantation phänotypisch heilbar ist, stellte sich die Frage, ob eine Gentherapie eine alternative Behandlungsstrategie darstellen könnte. Zur Prüfung der prinzipiellen Möglichkeit dieses Ansatzes untersuchten wir die Wirksamkeit des adenoviralen Transfers des Morbus-Wilson-Gens in der Long-Evans-Cinnamon-Ratte (LEC), einem etablierten Morbus-Wilson-Tiermodell.
Methoden: Die Transduktionseffizienz wurde mit RT-PCR, Western blot und Immunfluoreszenz geprüft. Der therapeutische Effekt wurde mittels Ferrooxidaseaktivitätsassay und Holocoeruloplasmin-Messungen im Serum sowie Messung der biliären/intestinalen und renalen Kupferausscheidung analysiert. Mittels Positronenemmisionstomographie wurde der Einfluss auf die Kupferhomöostase untersucht.
Ergebnisse: Der erfolgreiche, jedoch nur vorrübergehende adenovirale Transfer des ATP7B-Gens war auf RNA- und Proteinebene deutlich erkennbar. Parallel hierzu zeigte sich der funktionelle Erfolg in der vorrübergehenden Wiederherstellung des Holocoeruloplasmins und seiner Ferrooxidaseaktivität im Serum sowie in der gesteigerten, biliären Kupferausscheidung.
Schlussfolgerung: Die Daten verdeutlichen die prinzipielle Anwendbarkeit der Gentherapie bei Morbus Wilson. Aufgrund des nur vorrübergehenden Therapieerfolgs des adenoviralen Gentransfers sind nun gentherapeutische Strategien mit höherer Transduktionseffizienz und längerandauerndem Therapieerfolg zu entwickeln.
Key words
Wilson Disease - Gene Therapy - Adenoviral Vector - LEC-Rat - PET
Schlüsselwörter
Morbus Wilson - Gentherapie - Adenoviraler Vektor - LEC-Ratte - PET
References
- 1
Wilson S AK.
Progressive lenticular degeneration: A familial nervous
disease associated with cirrhosis of the
liver.
Brain.
1912;
34
295-507
MissingFormLabel
- 2
Bull P C, Thomas G R, Rommens J M, Forbes J R, Cox D W.
The Wilson disease gene is a putative copper transporting
P-type ATPase similar to the Menkes gene.
Nat
Genet.
1993;
5
327-337
MissingFormLabel
- 3
Petrukhin K, Fischer S G, Piratsu M. et al .
Mapping, cloning and genetic characterization of the regions
containing the Wilson disease locus.
Nat
Genet.
1993;
5
338-343
MissingFormLabel
- 4
Tanzi R E, Petrukhin K, Chernov I. et al .
The Wilson disease gene is a copper transporting ATPase with
homology to the Menkes disease gene.
Nat
Genet.
1993;
5
344-350
MissingFormLabel
- 5
Duc H H, Hefter H, Stremmel W. et al .
His1069Gln and six novel Wilson disease mutations: Analysis
of relevance for early diagnosis and phenotype.
Eur J Hum
Genet.
1998;
6
613-623
MissingFormLabel
- 6
Stremmel W.
Wilson disease: Clinical presentation, treatment, and
survival.
Ann Intern
Med.
1991;
115
720-726
MissingFormLabel
- 7
Scheinberg I H, Sternlieb I, Schilsky M, Stockert R J.
Penicillamine may detoxify copper in Wilson’s
disease.
Lancet.
1987;
2
95
MissingFormLabel
- 8
Smolarek C, Stremmel W.
Therapy of Wilson disease (author’s
transl.).
Z
Gastroenterol.
1999;
37
293-300
MissingFormLabel
- 9
Brewer G J, Yuzbasiyan-Gurkan V, Young A B.
Treatment of Wilson’s disease.
Semin
Neurol.
1987;
7
209-220
MissingFormLabel
- 10
Walshe J M.
Penicillamine: A new oral therapy for Wilson’s
disease.
Am J
Med.
1956;
2
487-495
MissingFormLabel
- 11
Schumacher G, Platz K P, Mueller A R. et al .
Liver transplantation: Treatment of choice for hepatic and
neurological manifestation of Wilson’s disease.
Clin
Transplant.
1997;
11
217-224
MissingFormLabel
- 12
Khanna A, Jain A, Eghtesad B, Rakela J.
Liver transplantation for metabolic liver
diseases.
Surg Clin North
Am.
1999;
79
153-162
MissingFormLabel
- 13
Schumacher G, Mueller A R, Platz K P. et al .
Neurologic symptoms improve in patients with Wilson’s
disease despite immunosuppression.
Transplant
Proc.
1996;
28
3099-3100
MissingFormLabel
- 14
Chen C L, Chen Y S, Lui C C, Hsu S P.
Neurological improvement of Wilson’s disease after
liver transplantation.
Transplant
Proc.
1997;
29
497-498
MissingFormLabel
- 15
Kassam N, Witt N, Kneteman N, Bain V G.
Liver transplantation for neuropsychiatric Wilson
disease.
Can J
Gastroenterol.
1998;
12
65-68
MissingFormLabel
- 16
Bax R T, Hassler A, Luck W. et al .
Cerebral manifestation of Wilson’s disease successfully
treated with liver
transplantation.
Neurology.
1998;
51
863-865
MissingFormLabel
- 17
Lui C C, Chen C L, Cheng Y F, Lee T Y.
Changes in neuroimaging in Wilson’s disease following
orthotopic liver transplantation.
Transplant
Proc.
1998;
30
3324-3325
MissingFormLabel
- 18
Stracciari A, Tempestini A, Borghi A, Guarino M.
Effect of liver transplantation on neirological manifestation
in Wilson disease.
Arch
Neurol.
2000;
57
384-386
MissingFormLabel
- 19
Schilsky M, Scheinberg I, Sternlieb I.
Liver transplantation for Wilson’s disease: Indications
and
outcome.
Hepatology.
1994;
19
583-587
MissingFormLabel
- 20
Schilsky M, Stockert R J, Sternlieb I.
Pleiotropic effect of LEC mutation: A rodent model of
Wilson’s disease.
Am J
Physiol.
1994;
266
907-913
MissingFormLabel
- 21
Wu J, Forbes J R, Chen H S, Cox D W.
The LEC rat has a deletion in the copper transporting ATPase
gene homologous to the Wilson disease gene.
Nat
Genet.
1994;
7
541-545
MissingFormLabel
- 22
Yoshida M C, Masuda R, Sasaki M. et al .
New mutation causing hereditary hepatitis in the laboratory
rat.
J
Hered.
1987;
78
361-365
MissingFormLabel
- 23 Mori M, Yoshida M C, Takeichi N, Taniguchi N. ”The LEC
rat”. Tokio; Springer-Verlag 1991
MissingFormLabel
- 24
Löser P, Sandig V, Kirillova I, Strauss M.
Evaluation of HBV promotors for use in hepatic gene
therapie.
Biol Chem
Hoppe-Seyler.
1996;
377
187-193
MissingFormLabel
- 25
Chartier C, Degryse E, Gantzer M. et al .
Efficient generation of recombinant adenovirus vectors by
homologous recombination in Escherichia coli.
J
Virol.
1996;
70
4805-4810
MissingFormLabel
- 26
Sandig V, Löser P, Lieber A, Kay M A, Strauss M.
HBV-derived promotors direct liver-specific expression of an
adenovirally transduced LDL receptor gene.
Gene
Ther.
1996;
3
1002-1009
MissingFormLabel
- 27
Terada K, Nakako T, Yang X L. et al .
Restoration of holoceruloplasmin synthesis in LEC rat after
infusion of recombinant adenovirus bearing WND cDNA.
J Biol
Chem.
1988;
237
1815-1820
MissingFormLabel
- 28
Laemmli U K.
Cleavage of structural proteins during the assembly of the
head of bacteriophage
T4.
Nature.
1970;
227
680-685
MissingFormLabel
- 29
Schaefer M, Hopkins R G, Failla M L, Gitlin J D.
Hepatocyte-specific localization and copper-dependent
trafficking of the Wilson’s disease protein in the liver.
Am J
of Physiol
Soc.
1999;
276
G639-G646
MissingFormLabel
- 30
Sato M, Schilsky M L, Stockert R J, Morell A G, Sternlieb I.
Detection of multiple forms of human
ceruloplasmin.
J Biol
Chem.
1990;
5
2533-2537
MissingFormLabel
- 31
Schosinsky K H, Lehmann H P, Beeler M F.
Measurement of ceruloplasmin from its oxidase activity in
serum by use of o-dianisidine dihydrochloride.
Clin
Chem.
1974;
20
1556-1563
MissingFormLabel
- 32
Holte S, Ostertag H, Kesselberg M.
A preliminary evaluation of a dual crystal positron
camera.
J Comput Assist
Tomogr.
1987;
11
691-697
MissingFormLabel
- 33
Yamada T, Agui T, Suzuki Y, Sato M, Matsumoto K.
Inhibition of the copper incorporation into ceruloplasmin
leads to the deficiency in serum ceruloplasmin activity in Long-Evans Cinnamon
mutant rat.
J Biol
Chem.
1993;
268
8965-8971
MissingFormLabel
- 34
Holtzman N A, Gaumnitz B M.
Studies on the rate of release and turnover of ceruloplasmin
and apoceruloplasmin in rat plasma.
J Biol
Chem.
1970;
245
2354-2358
MissingFormLabel
- 35
Wesch H, Przuntek H, Feist D.
Wilson’ disease: A rapid diagnosis and differentiation
of heterozygous and homozygous carriers with 64CuCl2 (author’s
transl).
Dtsch Med
Wochenschr.
1980;
105
483-488
MissingFormLabel
- 36
Klein D, Lichtmannegger J, Heinzmann U, Summer K H.
Dissolution of copper-rich granules in hepatic lysosomes by
D-penicillamine prevents the development of fulminant hepatitis in Long-Evans
cinnamon rats.
J
Hepatol.
2000;
32
193-201
MissingFormLabel
- 37
Park F, Ohashi K, Chiu W, Naldini L, Kay M A.
Efficient lentiviral transduction of liver requires cell
cycling in vivo.
Nat
Genet.
2000;
24
49-52
MissingFormLabel
- 38
Palombo F, Monciotti A, Recchia A. et al .
Site-specific integration in mammalian cells mediated by a
new hybrid Baculovirus-Adeno-associated virus vector.
J
Virol.
1998;
72
5025-5034
MissingFormLabel
- 39
Recchia A, Parks R J, Lamartina S. et al .
Site-specific integration mediated by a hybrid
adenovirus/adenoassociated virus vector.
Proc Natl Acad Sci
USA.
1999;
96
2615-2620
MissingFormLabel
- 40
Hofman C, Löser P, Cichon G. et al .
Ovine adenovirus vectors overcome preexisting humoral
immunity against human adenoviruses in
vivo.
J Virol.
1999;
73
6930-6936
MissingFormLabel
- 41
Overturf K, Al-Dhalimy M, Tanguay R. et al .
Hepatocytes corrected by gene therapy are selected in vivo in
a murine model of hereditary tyrosinaemia type I.
Nat
Genet.
1996;
12
266-273
MissingFormLabel
Korrespondenzanschrift
Wolfgang Stremmel, MD
Medizinische Klinik IV
Klinikum der Universität
Heidelberg
Bergheimer Straße 58
69115 Heidelberg
eMail: wolfgang_stremmel@med.uni-heidelberg.de