Division of Human Gene Therapy, University of Alabama at Birmingham, Room 614, Wallace Tumor Institute, 1824 Sixth Avenue South, Birmingham, AL 35294, USA1
Author for correspondence: Ramon Alemany. Fax +1 205 975 7949. e-mail ramon.alemany{at}ccc.uab.edu
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Different viruses are cleared from the blood by Kupffer cells (KC) (Kirn et al., 1982 ). Blockage of macrophages has indicated the interaction of adenovirus and KC (Lieber et al., 1997
; Wolff et al., 1997
; Worgall et al., 1997
). When KC are not blocked, 90% of the adenovirus genome is cleared from the liver in 24 h (Worgall et al., 1997
). To demonstrate the direct interaction of adenovirus with KC, we injected Ad-CMV-LacZ virus (Alemany et al., 1996
) labelled with red fluorophore (Cy3, Amersham; Leopold et al., 1998
) into the tail vein of BALB/c mice (1011 virions per mouse). Ten minutes later, we injected fluorescein-labelled latex beads (2 µm diameter; Sigma) as a KC tracer. After a further 10 min, livers were resected and embedded for cryosection. The co-localization of virus and latex beads within KC was readily observed as patchy aggregates all over the sinusoids (Fig. 1C
). In most instances, co-localization was evident as yellow fluorescent regions containing several latex beads. Some red fluorescence was also observed with no beads, probably due to incomplete marking of all KC. In order to confirm further the role of KC in virus uptake, KC were depleted with GdCl3 (three tail-vein injections of 200 µl of a 2 mg/ml solution at 54, 30 and 6 h before injection of adenovirus and latex beads). The distribution of adenovirus in the liver changed from the patchy pattern to a regular, diffuse pattern enriched towards the perivenous region of the liver (Fig. 1D
). Whether this periportal to perivenous enrichment reflects differences in the levels of coxsackievirus/adenovirus receptor (CAR) in parenchymal or endothelial cells or the effect of other factors requires further investigation. The patchy distribution of adenovirus capsids taken up by KC reflects the main deposition site of the inoculum. More sensitive techniques to detect viral capsid proteins would probably reveal the distribution of smaller amounts of virus in other cell types. An attempt has recently been made to quantify the distribution of an adenovirus vector among different liver cell types after intravenous injection in rats (Davern et al., 1999
). Although hepatocytes, stellate cells, KC and endothelial cells are transduced, the measurements of gene expression probably do not reflect the amount of virus adsorption. Indirect evidence that each liver cell type interacts with adenovirus comes from studies of liver toxicity (Lieber et al., 1997
).
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The mechanism of adenovirus uptake by KC or other macrophages is not known. Human alveolar macrophages are not transduced efficiently with adenovirus vectors due to low CAR levels, although transduction is blocked by soluble fibre (Kaner et al., 1999 ). Monocytes and their derived macrophages also express adenovirus transgenes very inefficiently (Huang et al., 1996
). Attachment and expression in this case are not blocked by soluble fibre. Adenovirus entry into alveolar macrophages by a non-CAR pathway could lead to virus degradation rather than transgene expression. From the point of view of clearance, these scavenger pathways are most important. KC clear bacteria, parasites, colloids, erythrocytes and any particulate material in circulation (such as latex beads) (Steffan, 1997
). Size and net charge are major determinants of clearance, and it has been noted that the charge of the adenovirus capsid may play a decisive role in tropism (Mei & Wadell, 1995
). Interestingly, the neutral mouse adenovirus is adapted to circulate in blood and has a tropism for endothelial cells. To study the effect of Ad5 charge on blood clearance, we deleted 13 residues (EEEDDDNEDEVDE) of the hexon hypervariable region 1 (HVR1) (Crawford-Miksza & Schnurr, 1996
). HVR1 contains the largest number of charged residues that differ among serotypes. We used the oligonucleotide 5' ctgctcttgaaataaacctaCAAGCTGAGCAGCAAAAAACTC 3' (capital letters indicate residues after the deletion point) for site-directed mutagenesis (Transformer; Clontech) in a plasmid containing a portion of the hexon gene (XmaI fragment, bp 1891820373 of Ad5). The mutated XmaI fragment was inserted into the plasmid containing the GFP cassette in E3 described above. Virus AdhN was generated by transfection of 293 cells and purified as above. The deletion was confirmed by DNA sequencing. Zeta potential charge analysis (PALS analyser) of a solution containing 1012 virus particles/ml PBS (pH 7·4) of Adwt gave a value of -9·76±0·98 mV compared with -6·28±0·62 mV for AdhN, indicating a substantial charge modification (three independent measurements). When AdhN was injected into the vena cava of three BALB/c mice, we did not observe any significant delay in virus clearance (Fig. 2
). A more laborious analysis is warranted to rule out the possibility that charge does not affect clearance, because most other negative residues are scattered throughout the hexon and fibre sequences. An alternative approach would be to compare the clearance of different adenovirus serotypes.
Coating with PEG (PEGylation) is commonly used to avoid protein clearance by KC and other macrophages. PEGylated stealth liposomes and nanoparticles show increased blood persistence (Allemann et al., 1995 ; Gabizon et al., 1994
). Adenovirus has been PEGylated to avoid neutralization by antibodies (ORiordan et al., 1999
). Due to the potential use of PEGylated adenoviruses as gene-delivery vehicles, we studied their blood clearance rate. Tresyl-monomethoxypolyethylene glycol (TMPEG) from PolyMASC Pharmaceuticals was obtained from Fluka. Adwt (2x1012 virus particles/ml in PBS) was diluted 2-fold with 130 mM sodium phosphate pH 7, 5% sucrose, and 0·1 ml was added to 4·1 mg TMPEG (i.e. 4·1%, w/v). After 30 min incubation at room temperature, virus was injected into the vena cava of mice. Infectivity of the PEGylated virus was measured by serial dilution and infection of A549 cells. In agreement with previous reports (Chillon et al., 1998
), we found a 300- to 1000-fold loss of infectivity with 4·1% TMPEG and a proportionally smaller reduction when using less TMPEG (not shown). The clearance rate during the first 30 min gave K=0·02, 4-fold slower than the non-PEGylated virus (Fig. 2B
).
In summary, adenovirus clearance from blood results in a virus half-life of less than 2 min. Similar to other non-blood-borne viruses, adenovirus is efficiently phagocytosed by KC and the blood clearance kinetics suggest a high capacity, non-specific uptake. Adenovirus clearance is not affected by partial charge neutralization via deletion of the hexon acidic stretch. More extensive charge depletion, or other modifications of the viral capsid such as elimination of CAR binding and integrin binding, need to be evaluated as ways of increasing blood persistence. Finally, it must also be noted that, whereas murine models are most commonly used in preclinical gene therapy research, clearance studies in other animal species are needed to allow generalization from these results.
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References |
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Received 28 April 2000;
accepted 24 July 2000.