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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 8, 2001 as doi:10.1096/fj.00-0892fje. |
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* U.129 INSERM, ICGM, 75014 Paris, France;
Service de Réanimation Polyvalente, Hôpital Cochin, 75014 Paris, France;
Laboratoire d’Anatomie Pathologique, Hôpital Bicêtre, 94275 Le Kremlin-Bicêtre, France; and
UMR1582, CNRS-IGR-Aventis, Institut Gustave-Roussy 94805 Villejuif, France
2Correspondence: U.129 INSERM, ICGM, 24 Rue du Faubourg St. Jacques, 75014 Paris, France. E-mail: gilgenkrantz{at}cochin.inserm.fr
SPECIFIC AIMS
Stable liver gene transfer is generally limited by the low efficiency of commonly used vectors. One way to circumvent this difficulty is to confer a selective advantage on transduced hepatocytes, allowing them to progressively repopulate the liver. The aim of this study was to investigate the possibility to repopulate a normal liver to a desired level with in vivo retrovirally engineered hepatocytes.
PRINCIPAL FINDINGS
1. A retroviral vector encoding Bcl-2 can repopulate the liver up
to 85% after 10 wk
We have previously shown that transplanted transgenic hepatocytes
expressing Bcl-2 could repopulate a normal mouse liver submitted to
repeated Fas apoptotic challenges.
We constructed two retroviral plasmids containing either the green fluorescent protein reporter gene alone (MFG-GFP) or a bicistronic cassette including human Bcl-2 cDNA and the GFP reporter gene (MFG-BIG) separated by an internal ribosomal entry site. To compare our liver repopulation approach with direct retroviral transduction, one group of C57Bl/6 mice was injected with MFG-BIG retrovirus (n=11) and another with MFG-GFP (n=8) retrovirus. The basal level of retroviral transduction was determined in a sample of animals (n=4 for each group) killed before any subsequent treatment. To confer a survival advantage on transduced hepatocytes expressing Bcl-2, the remaining animals were submitted to 10 weekly apoptotic challenges using the Fas pathway agonist antibody JO2. A subgroup of MFG-BIG-injected mice (n=3) not submitted to JO2 and killed 10 wk after retroviral injection served as controls. Bcl-2 and GFP immunohistochemistry on liver sections before any JO2 treatment showed that transduced hepatocytes were found homogeneously scattered throughout the hepatic lobes, mostly in the midzonal region. Around 1% of hepatocytes was initially transduced in both MFG-BIG and MFG-GFP groups (1.45%±0.45 and 1.1%±0.24, respectively). After 10 courses of JO2, the MFG-BIG-injected mice showed extensive liver repopulation consisting of confluent nodules of 10 to 30 cells, reaching a mean level of 77%, the best animal achieving 85%. In contrast, in the MFG-BIG group not submitted to JO2, Bcl-2-expressing cells remained isolated at a level equivalent to that obtained before apoptotic challenges (1.1%±0.32). Finally, very scarce hepatocytes stained positively for GFP in the MFG-GFP group treated with JO2 (0.25%±0.1). Histological examination of the livers either before or after 10 injections of JO2 antibody showed conserved lobular architecture. No dysplasia was detected after extensive analysis of all transduced and repopulated livers.
2. Bicistronic retroviral vector allows for expression of a
transgene of interest in nearly the entire liver
Expression of the second transgene was analyzed by macroscopic
analysis of livers under direct fluorescent microscopy. In the MFG-GFP
group submitted to JO2, no fluorescence could be detected. In contrast,
large fluorescent nodules were visualized in MFG-BIG-injected mice
submitted to JO2 treatment whereas hardly detectable pinpoint spots
were seen in MFG-BIG-injected mice not submitted to JO2. These results
were further confirmed by immunostaining of liver sections with
antibodies to GFP that showed confluent clusters of GFP-positive
hepatocytes in all liver lobes of the MFG-BIG group injected with JO2.
3. Time course study of liver repopulation demonstrates that liver
repopulation is highly predictable
To follow the progressive proliferation of cells carrying the
selective advantage gene and a decline in the number of hepatocytes
devoid of the selective advantage gene with the successive rounds of
apoptosis/regeneration, we coinjected mice with two retroviral vectors:
a ß-galactosidase-expressing retrovirus (MFG-LacZ) and MFG-BIG
retrovirus. Liver repopulation was assessed by immunohistochemistry
before any apoptotic challenge and after 2, 4, 6, and 8 weekly JO2
injections, showing the progressive expansion of Bcl-2-expressing cells
(Fig. 1
). We then measured the proportion of LacZ-expressing cells and observed
their progressive decline with JO2 (Fig. 2
). A control group of animals not submitted to JO2 and killed 8 wk after
retroviral transduction showed no statistical difference when compared
with animals killed before any apoptotic challenge regarding the number
of Bcl-2- and Lac-Z-expressing cells.
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We determined the theoretical kinetics of liver repopulation and Lac-Z
decline by a mathematical model presuming that 1)
Bcl-2-expressing cells and nontransduced hepatocytes had an equal
proliferation capacity and 2) only non-Bcl-2 hepatocytes
were sensitive to JO2, the Bcl-2 positive cells being totally protected
from apoptosis. The theoretical percentage of repopulation at the time
of each apoptotic challenge was calculated using the formula:
Xn = Xn-1 x
100/100-Y(100-Xn-1), where Y represents the
percentage of destroyed hepatocytes and ‘n’ the number of apoptotic
challenges. We had previously determined that an injection of 0.12
mg/kg of JO2 led to a cytolysis ranging from 40% to 47%. Experimental
data regarding the increase in Bcl-2-expressing cells was not
statistically different from the theoretical curves (Fig. 2A
). Theoretical percentages of Lac-Z cells were calculated
using the formula Zn =
Zn-1(1-Y) x
100/100-Y(100-Xn-1), where Y represents the
percentage of destroyed hepatocytes, ‘n’ the number of apoptotic
challenges, and X the percentage of Bcl-2-expressing hepatocytes
previously determined. Again, the decline in cells expressing only LacZ
was not statistically different from the theoretical curves (Fig. 2B
).
CONCLUSIONS
Liver gene therapy has been limited by the low efficiency of available gene transfer vectors to stably integrate a therapeutic transgene in a large proportion of hepatocytes. Moreover, human clinical trials would require production of high titers of these vectors, which until now could hardly have been achieved. One way to circumvent these difficulties is to selectively amplify transduced hepatocytes directly in vivo.
We have previously shown that transgenic hepatocytes expressing Bcl-2 could repopulate a normal mouse liver submitted to repeated Fas apoptotic challenges. Based on this approach, we have developed the MFG-BIG retroviral vector encoding both Bcl-2 and a reporter gene. Here we demonstrate that as few as 1.45% of initially in vivo retrovirally engineered hepatocytes can repopulate up to 85% of the liver after 10 weekly injections of the Fas agonist antibody. This high level of positive hepatocytes was obtained only in MFG-BIG treated animals and not in control groups. These results confirmed that liver repopulation was due to selection of Bcl-2-transduced cells rather than to high efficiency of the initial gene transfer or to spontaneous proliferation of transduced hepatocytes in the absence of JO2 treatment.
An efficient retroviral repopulation approach has already been reported in a murine model of hereditary tyrosinemia type I. However, contrary to our approach, these results have been obtained in a specific diseased liver with a constitutive turnover of resident hepatocytes and therefore cannot be transposed to another mouse model. Moreover, our bicistronic retroviral vector allows us to express another transgene in a very high proportion of hepatocytes, which could have real therapeutic potential. We were able to design a mathematical model of our repopulation strategy that allowed us to tightly predict the number of JO2 challenges necessary to achieve the desired level of liver repopulation. This model allows one to obtain a genetically modified liver expressing a gene of interest in a desired proportion of hepatocytes and could help in determining the proportion of liver repopulation needed to correct a phenotype in a mouse model of a human disorder.
The possibility of repopulating the liver could have important applications in the treatment of various human genetic diseases such as deficiencies in circulating proteins. Acquired disorders could also benefit from this strategy—for example, chronic hepatitis—by repopulation of the liver with cells expressing antiviral protein(s) or antisense RNA(s) or hypercholesterolemia and atherosclerosis by promoting high-level secretion of anti-atheromatous apolipoproteins. However, the Bcl-2/JO2 approach used here will probably not be applicable in humans, and new strategies able to confer a selective advantage on transduced hepatocytes will be needed. These strategies should either protect transduced hepatocytes from a hepatotoxic drug that is safer and easier to use than Fas agonists or selectively stimulate their regeneration capacity, as has recently been shown with blood cells that were conditionally expanded from a small number of retrovirally transduced bone marrow cells. In fact, a similar strategy of tissue repopulation by engineered cells recently led to the successful treatment of young patients with hereditary X-linked combined immunodeficiency. We are confident that in the future this type of ‘regenerative medicine’ will lead to therapeutic progress in various pathologies affecting organs capable of regeneration.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0892fje ; to
cite this article, use FASEB J. (June 8, 2001) 10.1096/fj.00-0892fje 
This article has been cited by other articles:
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  C. Mitchell, V. O. Mallet, J. E. Guidotti, C. Goulenok, A. Kahn, and H. Gilgenkrantz Liver Repopulation by Bcl-xL Transgenic Hepatocytes Am. J. Pathol., January 1, 2002; 160(1): 31 - 35. [Abstract] [Full Text] [PDF]
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