Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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To achieve efficient systemic gene delivery to the lung
with minimal toxicity, a vector was developed by chemically conjugating a cationic polymer, polyethylenimine (PEI), with anti-platelet endothelial cell adhesion molecule (PECAM) antibody (Ab). Transfection of mouse lung endothelial cells with a plasmid expression vector with
cDNA to luciferase (pCMVL) complexed with anti-PECAM Ab-PEI conjugate
was more efficient than that with PEI-pCMVL complexes. Furthermore, the
anti-PECAM Ab-PEI conjugate mediated efficient transfection at lower
charge plus-to-minus ratios. Conjugation of PEI with a
control IgG (hamster IgG) did not enhance transfection of mouse lung
endothelial cells, suggesting that the cellular uptake of anti-PECAM
Ab-PEI-DNA complexes and subsequent gene expression were governed by a
receptor-mediated process rather than by a nonspecific charge
interaction. Conjugation of PEI with anti-PECAM Ab also led to
significant improvement in lung gene transfer to intact mice after
intravenous administration. The increase in lung transfection was
associated with a decrease compared with PEI-pCMVL with respect to
circulating proinflammatory cytokine (tumor necrosis factor-)
levels. These results indicate that targeted gene delivery
to the lung endothelium is an effective strategy to enhance gene
delivery to the pulmonary circulation while simultaneously reducing toxicity.
cationic polymer; plasmid deoxyribonucleic acid; gene transfer; targeting; toxicity
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INTRODUCTION |
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GENE DELIVERY TO THE LUNG via intravenous
administration may be useful in treating pulmonary tumor metastases,
pulmonary hypertension, and acute respiratory distress syndrome (8, 30,
44). With a better understanding of the pathophysiology of pulmonary
diseases and the discovery of many therapeutic genes to correct the
diseased phenotype, the success of lung gene therapy is largely
dependent on the development of a vector or vehicle that can
efficiently deliver a gene to the lung. Adenoviral vectors are
relatively inefficient in gene transfer to the intact pulmonary
circulation as a result of either brief transit times (34) or lack of
receptors for uptake of adenoviruses on the endothelium (43). Recently, empirical optimization of nonviral vectors has led to the development of several lipid (or polymer)-based vectors that are highly efficient in transfecting lungs on intravenous injection (4, 11, 13-15, 18-23, 25, 36, 37, 39, 41, 45). Lung transfection via these
vectors is mainly attributed to their physical properties that give
rise to prolonged retention in the pulmonary circulation and efficient
interactions with the pulmonary vasculature (20, 25, 37). In this
study, we show that transfection efficiency of these vectors can be
enhanced by inclusion of a targeting ligand against platelet
endothelial cell adhesion molecule-1 (PECAM-1 or CD31). In addition,
the improved vector is associated with a decrease compared with
polyethylenimine (PEI)-firefly luciferase gene under the control of a
human cytomegalovirus immediate-early promoter (pCMVL) with respect to
circulating proinflammatory cytokine [tumor necrosis factor
(TNF)-] levels. Finally, dexamethasone (Dex) can further
improve the persistence of gene expression, suggesting that development
of an endothelium-specific vector together with the use of an
immunosuppressant is an effective approach for targeted gene delivery
to the lung via systemic administration.
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METHODS AND MATERIALS |
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Chemicals. Rat monoclonal antibody (Ab) against mouse PECAM-1 was purchased from PharMingen (San Diego, CA). Hamster IgG was purchased from Sigma (St. Louis, MO). Branched PEIs of different molecular masses (25, 60-70, or 750 kDa) were from Aldrich (Milwaukee, WI). Linear PEI of 22 kDa (Exgene 500) was obtained from Euromedex (Souffleweyersheim, France). N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and dithiothreitol were from Pierce (Rockford, IL). Na125I was from DuPont NEN (Boston, MA). Luciferase assay kit was obtained from Promega (Madison, WI). All other chemicals were of reagent grade.
Mice. CD-1 mice (16-18 g, female) were from Charles River Laboratories (Wilmington, MA). Animals were kept at University Central Animal Facility. All experiments were conducted with protocols approved by the Institutional Animal Care and Use Committee.
Purification and iodination of plasmid DNA. Plasmid that
contains cDNA of firefly luciferase driven by CMV promoter was
amplified in the DH5 strain of Escherichia coli and then
isolated by alkaline lysis and purified by cesium chloride
centrifugation (33). Endotoxin was further removed from the DNA by
being passed through a QIAGEN-tip 500 column (QIAGEN, Valencia, CA).
The endotoxin level in the plasmid used in this study was <0.01
endotoxin unit/mg DNA. Plasmid DNA was labeled with 125I
with a published method (32) and purified by a spin column (Bio-Spin-P30). The purified 125I-labeled DNA contained
roughly equal amounts of supercoiled and relaxed closed circular DNA as
examined by gel electrophoresis and autoradiography.
Preparation of Ab-PEI conjugates. To a solution of 1 mg of PEI (1 mg/ml in 10 mM HEPES, pH 7.4, and 0.25 M NaCl) was added a 6 molar excess of SPDP (1 mg/ml in dry N,N-dimethylformamide). After incubation at room temperature for 1 h, the SPDP-derivatized PEI was purified by being passed through a Sephadex G-25 column.
To a solution of 4 mg of anti-PECAM Ab (2 mg/ml in 10 mM HEPES, pH 7.4, and 0.25 M NaCl) was added 25 µg of SPDP (1 mg/ml in dry N,N-dimethylformamide). After incubation at room temperature for 1 h, the reaction mixture was applied to a Sephadex G-25 column equilibrated with 0.1 M acetate buffer, pH 4.5, and eluted with the same buffer. Dithiothreitol was added to the modified Ab solution at a final concentration of 50 mM. After incubation at room temperature for 30 min, the reaction mixture was applied to a Sephadex column equilibrated with 10 mM HEPES, pH 7.4, and 0.25 M NaCl and eluted with the same buffer. The purified sulfhydryl-containing Ab was immediately mixed with SPDP-derivatized PEI at a molar ratio of 2:1 (Ab to PEI). The mixture was stirred at 4°C overnight, and the conjugate was isolated by ion-exchange chromatography as described by Kircheis et al. (16). Finally, the conjugate was dialyzed against 2.5 mM HEPES for 48 h at 4°C to remove salts.
Similarly, a hamster IgG-PEI conjugate was also prepared and used as a control in this study.
Zeta potential analysis. The analysis of the charge on PEI-DNA
or Ab-PEI-DNA complexes was performed by examining its zeta potential
with a ZetaSizer 4 (Malvern Instruments, Southborough, MA). The system
was calibrated with a 50-mV standard (DTS 50/50 Standard,
Malvern Instruments) as recommended by the manufacturer. Experimental
samples (3 ml) were measured six times for 30 s at 1,000 Hz with zero
field correction.
In vitro transfection of mouse lung endothelial cells. Cultured mouse lung endothelial cells (MLECs) were a gift from Dr. Mary Ellen Gerritsen (Genentech, San Francisco, CA). MLECs were isolated from endotoxin-treated C57BL/6 mice after collagenase digestion of the lung and selection for vascular cell adhesion molecule-1 expression by fluorescence-activated cell sorting (10). MLECs were cultured on gelatin-coated containers and used between passages 16 and 19. Medium contained fetal bovine serum (20%), endothelial cell growth factor (50 µg/ml), and heparin (100 U/ml) in Dulbecco's modified Eagle's medium. MLECs were plated on 48-well plates at a density of 2.5 × 104 cells/well. pCMVL complexed with PEI or Ab-PEI conjugate at different molar ratios of PEI nitrogen to DNA phosphate groups (N/P) was added at 1 µg/well in 500 µl of serum-free medium. Four hours after transfection, the medium was replaced with fresh complete medium, and the cells were incubated for another 48 h. Cells were then washed twice with cold PBS and lysed with 200 µl of lysis buffer for 3 min. Cell lysates were centrifuged at 14,000 g for 10 min, and 20 µl of the supernatant were analyzed for luciferase activity with an automated LB 953 luminometer equipped with an automated injector (Berthod, Bad Wildbad, Germany). Gene expression is expressed as relative light units per microgram of protein.
In vivo distribution of Ab-PEI-DNA complexes. 125I-labeled pCMVL complexed with PEI or Ab-PEI conjugates was injected into the tail vein of mice at a dose of 25 µg/mouse. At indicated times after injection, mice were killed. Major organs were collected and counted for 125I radioactivity. The result is expressed as the percentage of injected dose per organ. To evaluate whether pretreatment of mice with free Ab affected DNA distribution, mice received a tail vein injection of free Ab at a dose of 150 µg/mouse before injection of labeled complexes. The in vivo distribution of DNA in major organs was then similarly evaluated.
Gene expression in the lung after intravenous injection of Ab-PEI-DNA complexes. pCMVL was used as a reporter gene in this experiment. Groups of five mice received tail vein administration of DNA complexed with PEI or Ab-PEI conjugate. Twenty-four hours later, mice were bled from the retroorbital sinuses under anesthesia and killed by cervical dislocation. Lungs were collected and washed twice with cold saline. Lungs were homogenized with lysis buffer (0.05% Triton X-100, 2 mM EDTA, and 0.1 M Tris, pH 7.8) with a tissue tearor (Biospec Products, Bartlesville, OK). After two cycles of freeze and thaw, the homogenates were centrifuged at 14,000 g for 10 min at 4°C, and 20-µl aliquots of the supernatants were analyzed for luciferase activity. Gene expression is expressed as relative light units per milligram of protein. To evaluate whether pretreatment of mice with anti-PECAM Ab will affect lung transfection, mice received intravenous injections of free Ab at a dose of 150 µg/mouse before the injection of Ab-PEI-DNA complexes. Gene expression in the lung 24 h later was similarly evaluated.
Effect of Dex on lung transfection by Ab-PEI-DNA complexes. Dex was dissolved in PBS in a working solution of 1 mg/ml. To evaluate the effect of Dex on transgene expression, groups of four mice were given either Dex intraperitoneally at 100 µg/mouse for 3 days or PBS as a control beginning 1 day before intravenous administration of Ab-PEI-DNA complexes. At indicated times after injection, mice were killed, and gene expression in the lung was measured as described in Gene expression in the lung after intravenous injection of Ab-PEI-DNA complexes.
Immunoassays. Two hours after injection of Ab-PEI-DNA
complexes, mice were bled from the retroorbital sinuses under
anesthesia. The blood was allowed to stay at 4°C for 4 h and then
centrifuged at 14,000 g for 10 min at 4°C. Serum was
collected and kept at 80°C until used. Cytokine levels in
mouse serum were determined with a specific immunoassay kit for mouse
TNF-
(Genzyme, Cambridge, MA). Samples were analyzed in triplicate
on a plate reader, and cytokine levels were calculated by linear
regression analysis based on values obtained from a standard curve.
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RESULTS |
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Linear PEI but not branched PEIs mediated efficient lung gene
transfer. A recent study (11) reported that DNA complexed with
linear PEI can efficiently transfect lungs after intravenous administration. This result was confirmed in this study. The
transfection efficiency is closely related to N/P. Increasing N/P led
to a steady increase in transfection efficiency (Fig.
1). The increase in
transfection, however, is associated with an increase in toxicity. At
N/P of 16:1, piloerection was found in all injected mice, and one died
within 24 h after the injection. Interestingly, PEIs of branched
isomers are considerably less efficient in mediating lung gene transfer
at all N/P values examined. Moreover, branched PEIs, particularly those
of higher molecular masses, are more toxic than linear PEI (11). Thus
linear PEI was used in all subsequent studies.
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Conjugation with Ab led to shielding of surface charge in PEI-DNA
complexes. Zeta potential measurements are indicative of the
surface charge of polymer-DNA particles (Fig.
2). Nonconjugated PEI-DNA complexes had
zeta potentials ranging from strongly negative (35 mV; N/P = 1:1) to strongly positive (about +25 mV; N/P = 6:1 to
9:1). Conjugation with anti-PECAM Ab resulted
in a decrease in the surface charge of the complexes at N/P values of
6:1 to 9:1. At N/P of 1:1, the surface charge of Ab-conjugated
particles was similar to that of nonconjugated complexes. This might be due to the fact that the surface charge of particles is mainly contributed by the negatively charged plasmid DNA at low N/P values. Similar shielding effect was noticed when PEI was conjugated with the
control hamster IgG (data not shown).
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Anti-PECAM Ab-PEI-DNA complexes mediated efficient transfection of
MLECs. As shown in Fig.
3, PEI alone resulted in
transfection of MLECs. Transfection of MLECs by PEI-DNA complexes is
mainly due to the nonspecific charge interaction between the cells and the complexes because increasing the net positive charge of PEI-DNA complexes by increasing N/P values (from 1:1 to 6:1) led to significant increases in transfection efficiency. Conjugation of PEI with anti-PECAM Ab led to a significant improvement in MLEC transfection. More importantly, the anti-PECAM Ab-PEI conjugate mediated efficient MLEC transfection at a much lower N/P compared with unmodified PEI. At
N/P of 3:1, the level of transfection of MLECs by Ab-PEI-DNA complexes
was ~100-fold higher than that by PEI-DNA complexes. In contrast,
conjugation of a control IgG (normal hamster IgG) did not improve MLEC
transfection. These results suggest that transfection of MLECs by the
specific conjugate is largely governed by a receptor-mediated process
rather than by a nonspecific charge interaction.
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Prolonged retention of PEI-DNA complexes in lungs after conjugation
with anti-PECAM Ab. Figure 4 shows
uptake of DNA by lungs over time after intravenous administration of
PEI-pCMVL with and without anti-PECAM Ab. Five minutes after injection
of PEI-pCMVL, ~20% of the injected dose was retained in the lung.
Conjugation of PEI with anti-PECAM Ab resulted in a significant
increase in the lung uptake of plasmid DNA. Specificity of enhanced
uptake due to anti-PECAM Ab was confirmed by reducing the percentage of
lung retention of anti-PECAM Ab-PEI-DNA to that achieved by PEI-DNA
after pretreatment of mice with free anti-PECAM Ab. Figure 5 shows the distribution of PEI-DNA
complexes in several major organs before and after conjugation with
anti-PECAM Ab. Conjugation of PEI with Ab also significantly increased
DNA uptake by the heart, although the absolute amount of DNA in the
heart was much less than in the lung. In addition, the increase in DNA
uptake in the lung and heart after conjugation with anti-PECAM Ab was associated with a decrease in the uptake of DNA in the liver. This
change in the distribution was reversed when mice were pretreated with
free anti-PECAM Ab. Conjugation of PEI with a control IgG (hamster IgG)
did not improve lung uptake of DNA (data not shown). These results
suggest that the improved uptake of PEI-DNA complexes in the lung after
conjugation with anti-PECAM Ab is attributed to the specific
interaction of complexes with the pulmonary endothelium.
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Improved lung transfection by PEI-DNA complexes after conjugation
with anti-PECAM Ab. Figure
6 shows lung
transfection by PEI-DNA complexes before and after conjugation with
anti-PECAM Ab. Intravenous administration of PEI-DNA complexes gave a
moderate level of gene expression in the lungs. Conjugation of PEI with anti-PECAM Ab led to an ~20-fold increase in the level of gene expression. The improvement in gene expression was reduced when mice
were pretreated with free anti-PECAM Ab. Conjugation of PEI with a
control IgG (hamster IgG) was not associated with an increase in gene
expression relative to PEI-DNA, further supporting the specificity of
the targeting ligand (anti-PECAM Ab).
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Reduced cytokine induction after conjugation with anti-PECAM
Ab. Li et al. (21) have recently shown that
intravenous administration of plasmid DNA complexed with cationic
lipids can trigger production of proinflammatory cytokines, which are
toxic to the treated animals and inhibit transgene expression. In this
study, serum levels of TNF- were also investigated after intravenous
injection of PEI-DNA complexes. The serum was collected 2 h after the
injection because TNF-
peaked at this time point (21, 38). As shown in Fig. 7, intravenous administration of
PEI-DNA also induced TNF-
production. Conjugation of PEI with
anti-PECAM Ab was associated with two- to threefold less induction of
TNF-
than that with PEI-DNA. The decrease in TNF-
was partially
reversible by pretreating mice with Ab to PECAM. Conjugation of PEI
with a control IgG also led to decreased TNF-
induction but to a
lesser extent.
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Dex enhanced lung gene transfer by anti-PECAM-PEI-DNA
complexes. With intravenous gene delivery via
lipid-protamine-DNA, Tan et al. (38) have recently shown
that Dex can not only improve the persistence of gene expression in
lungs but also shorten the refractory period for repeated dosing. Here
we show that lung transfection via anti-PECAM Ab-PEI-DNA can also be
improved by Dex. As shown in Fig. 8, the
levels of gene expression in Dex-treated groups were consistently
higher than those in PBS-treated groups at all time points examined.
Dex could also improve lung transfection when PEI-DNA complexes were
used (data not shown).
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DISCUSSION |
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An ideal gene delivery vector should efficiently deliver a gene to a target in a tissue-specific manner with minimal toxicity after intravenous administration. To achieve this goal, the vector should be stable in the blood circulation and efficiently escape recognition by cells of the reticuloendothelial system (RES). However, the requirement of the vector for targeted gene delivery to the pulmonary endothelium is less stringent than that of the vector for gene delivery to other tissues such as the liver due to the following two reasons: 1) endothelia are directly exposed to blood circulation and, therefore, are readily accessible for interactions with the vector, and 2) the lungs contain ~30% of the endothelial cells in the body, they receive the entire cardiac blood output, and they are the first capillary bed the vector will encounter after tail vein administration (28). Thus there is sufficient interaction of the vector with the pulmonary endothelium. Targeted delivery of chemotherapeutic drugs and enzymes to the lung has been successfully demonstrated with immunoliposomes (24, 26) and Ab-protein conjugates (2, 27, 28). Specific delivery of a gene to the pulmonary endothelium has also been demonstrated with an anti-thrombomodulin Ab-polylysine conjugate (40). The latter conjugate, although efficient in delivering a gene to the lung, could not give rise to an efficient gene expression. This is probably due to the poor internalization of thrombomodulin in endothelial cells in vivo (Huang, unpublished data). In addition, DNA complexed with Ab-polylysine conjugate cannot efficiently escape from the endosome due to the lack of an endosome-disruption mechanism.
Unlike polylysine, PEI can facilitate endosomal release and mediate efficient cell transfection in vitro without the use of an endosome lytic agent (6). Recently, it has been shown (1, 5, 7, 9, 11) that PEI is also a good vehicle for polynucleotide delivery in vivo after lung instillation, kidney perfusion, intracerebral injection, or systemic delivery to the lung or the liver. Interestingly, among PEIs of different molecular masses and geometrical isomers (branched or linear skeleton), only the linear PEI can bring about efficient lung transfection (Fig. 1). This is not likely to be due to the difference in the ability of these PEIs to mediate endothelial cell transfection because no significant difference was noticed in the transfection of cultured MLECs regardless of which PEI was used (data not shown). It is possible that DNA complexed with these PEIs may form different structures and interact with serum differently after intravenous administration.
Anti-PECAM Ab was chosen in this study as a targeting ligand to further enhance the transfection efficiency of PEI-DNA complexes. PECAM is a transmembrane adhesion molecule expressed at high levels on endothelial cells (>1 million copies/cell) that plays an important role in transendothelial migration of leukocytes (29). Unlike angiotensin-converting enzyme, PECAM expression levels are not markedly altered by cytokines (29). Anti-PECAM Ab-mediated pulmonary targeting has been well demonstrated in recent studies by Muzykantov et al. (28) and Gow et al. (12) with Ab-avidin conjugates. Results from this study clearly suggest that anti-PECAM Ab can also mediate efficient gene transfer to pulmonary endothelial cells in vitro and in vivo. As shown in Fig. 3, transfection of MLECs by Ab-PEI-DNA complexes was significantly higher than that by the corresponding PEI-DNA complexes. More importantly, the Ab-PEI-DNA complexes mediated efficient MLEC transfection at much lower plus-to-minus (N/P) ratios, suggesting that cellular uptake of DNA and the subsequent gene expression are governed by a receptor-mediated process rather than by the nonspecific charge interaction between MLECs and the PEI-DNA complexes. Conjugation of PEI with anti-PECAM Ab also significantly improved the efficiency of lung gene transfer in vivo (Fig. 6). The improved lung transfection is mainly attributed to the enhanced uptake of DNA in the lung because decreasing the DNA uptake by Ab pretreatment also led to decreased transfection in the lung. These results together with the finding that conjugation of hamster IgG did not give rise to any increase in either DNA uptake or transfection efficiency further indicate that improved lung transfection by Ab-PEI conjugates is also mediated by the specific anti-PECAM Ab.
Our group and others have recently reported that lungs can also be efficiently transfected via systemic administration of cationic lipidic vectors (4, 11, 13-15, 18-23, 25, 36, 37, 39, 41, 45). Gene transfer to the lung via these vectors is primarily dependent on their physical properties, including a net positively charged surface and a relatively rigid bilayer (15). After intravenous injection, these vectors rapidly interact with negatively charged blood components, resulting in formation of aggregates (19, 20, 25). Given a suitable lipid composition, these aggregates can be entrapped in the pulmonary vasculature for a prolonged period of time, allowing for a sufficient interaction with endothelial cells (20, 37). Linear PEI-DNA complexes might share a similar mechanism in lung transfection. Although efficient in gene delivery to lungs, intravenous administration of these vectors is associated with induction of large amounts of proinflammatory cytokines, which are not only toxic to animals but also cause rapid inactivation of gene expression and refractoriness to repeated dosing at frequent intervals (21). Recent studies (3, 17, 21, 35, 38) suggest that cytokine induction is mainly due to the nonspecific uptake of lipid-DNA complexes by immune cells and is largely mediated by the unmethylated CpG sequences in plasmid DNA. One approach we are currently pursuing is to mutate these potent CpG sequences in plasmid DNA without affecting gene expression. Other ways to decrease the toxicity are to improve vectors so that they will have minimal interactions with immune cells, which are likely the major source of cytokines. Results from this study suggest that the development of endothelium-specific vectors represents such an effective approach. Conjugation of PEI with anti-PECAM Ab not only brought about improved lung transfection (Fig. 6) but also led to a further reduction in cytokine levels (Fig. 7). Decrease in cytokine induction is probably due to the improved uptake of DNA by the lungs (Fig. 4), with a concomitant decrease in liver uptake (Fig. 5). This is in agreement with the finding of recent study (42) that the liver is the major source of cytokine production after intravenous injection of cationic lipidic vectors. The reduced cytokine production may also be related to the fact that the Ab-conjugated PEI has less of a tendency to aggregate than the native PEI after intravenous injection. As shown in Fig. 2, the surface charge of PEI-DNA complexes was shielded greatly after conjugation with an Ab. Such surface modification by an Ab will not only increase the specificity of PEI-DNA complexes toward endothelial cells but also render the complexes less likely to aggregate after exposure to serum. These effects could lead to decreased interaction with the RES and reduced cytokine induction.
Whereas the vector (including the plasmid DNA) can be improved to be more efficient in gene transfer and less inflammatory, another approach to further improve in vivo gene delivery is the use of an immunosuppressant. It has been shown (31) that the use of Dex improves adenovirus-mediated gene transfer to the lung. Tan et al. (38) have recently shown that Dex can not only prolong the persistence of gene expression after intravenous administration of lipid-protamine-DNA but also improve the efficiency of repeated dosing. Results from this study showed that lung gene transfer by Ab-PEI-DNA complexes was also significantly improved by Dex, supporting a potential therapeutic role of Dex in lung gene transfer with Ab-polymer conjugates.
In summary, an anti-PECAM Ab-PEI conjugate has been prepared that can mediate efficient lung gene transfer with reduced toxicity. The efficiency of gene transfer via this conjugate can be further enhanced by Dex. Future study is needed to incorporate into the conjugate a component such as polyethylene glycol that will decrease its interaction with the RES. This will further improve the specificity of the vector toward the pulmonary endothelium.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44935 and National Cancer Institute Grants CA-59327 and CA-71731 (to L. Huang); National Heart, Lung, and Blood Institute Grant HL-32154 (to B. R. Pitt); and a contract from Targeted Genetics (to L. Huang).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Li, Dept. of Pharmacology, Univ. of Pittsburgh School of Medicine, E1656 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: songli{at}prophet.pharm.pitt.edu).
Received 13 July 1999; accepted in final form 8 October 1999.
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