ARTICLE
doi:10.1006/mthe.2000.0201, available online at http://www.idealibrary.com on IDEAL
DNA/Dendrimer Complexes Mediate Gene Transfer into Murine Cardiac Transplants ex Vivo Yinong Wang,*,1 Peter Boros,* Jianhua Liu,* Lihui Qin,* Yalai Bai,* Anna U. Bielinska,† Jolanta F. Kukowska-Latallo,† James R. Baker, Jr.,† and Jonathan S. Bromberg*,2 *Institute for Gene Therapy and Molecular Medicine and the Recanati/Miller Transplantation Institute, Mount Sinai School of Medicine, New York, New York 10029 †Center for Biologic Nanotechnology, University of Michigan, Ann Arbor, Michigan 48105 Received for publication August 30, 2000; accepted in revised form October 3, 2000; published online November 16, 2000
Starburst polyamidoamine dendrimers are synthetic polymers with unique structural and physical characteristics suitable for DNA gene transfer. Our previous studies demonstrated that Starburst dendrimers augment plasmid-mediated gene transfer efficiency in a nonvascularized, cardiac transplantation model. In this study, the fifth generation of ethylenediamine core dendrimer was investigated for its ability to enhance gene transfer and expression in a clinically relevant murine vascularized heart transplantation model. The plasmid pMP6A-β-gal, encoding βgalactosidase (β-Gal), was incubated with dendrimers to form complexes. The complexes were perfused via the coronary arteries during donor graft harvesting, and reporter gene expression was determined by quantitative evaluation of X-Gal staining. The grafts infused with pMP6A-βgal/dendrimer complexes showed β-Gal expression in myocytes from 7 to 14 days. A number of variables for transfer of the DNA/dendrimer complexes were tested, including DNA:dendrimer charge ratios, concentrations of DNA and dendrimer, preservation solutions, ischemic time, and enhancement of vascular permeability by serotonin, papaverine, and VEGF administration. The results showed that DNA/dendrimer complexes containing 20 µg of DNA and 260 µg of dendrimer (1:20 charge ratio) in a total volume of 200 µl resulted in highest gene expression in the grafts. The results also showed that prolonged incubation (cold ischemic time) to 2 h and pretreatment with serotonin further enhanced gene expression. Key Words: gene transfer; dendrimer; plasmid DNA; transplantation.
INTRODUCTION Gene transfer into the heart holds therapeutic potential for several cardiovascular diseases and graft rejection, but efficient delivery of genetic material into myocytes and sustained high levels of expression of the transferred gene are required. Various strategies have been used to deliver genes to organs, and previous investigations have demonstrated that a variety of plasmid and viral vectors can introduce and express exogenous genes in transplants (1–8), thereby prolonging allograft survival by inhibiting alloantigen-specific immunity (9). However,
1Present address: Department of Surgery (Cardiothoracic), Yale University School of Medicine, 333 Cedar Street, 121 FMB, New Haven, CT 06510. 2To whom correspondence and reprint requests should be addressed at the Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1104, New York, NY 10029-6574. Fax: (212) 348-2474. E-mail:
[email protected].
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existing gene transfer systems, such as viral vectors (retroviral and adenoviral vectors) or nonviral vectors (plasmids, liposomes), pose problems because of mutagenesis, immunogenicity, inflammation, recombination, and targeting, in addition to problems with limited duration of gene expression. A class of synthetic, highly branched spherical polyamidoamine (PAMAM) dendrimer polymers has a structural advantage for gene transfer. These molecules are uniform in size with a high density of charged primary amino groups restricted to the surface and are highly soluble and stable in aqueous solution. Recent studies have shown that Starburst dendrimers are nonimmunogenic and can mediate the enhanced delivery of diverse nucleic acids, including single-stranded or double-stranded, natural or synthetic, and DNAs or RNAs (10, 11). Our recent study demonstrated that dendrimer significantly increased the efficiency of plasmid-mediated gene transfer and expression in murine, heterotopic, nonvascularized cardiac grafts. Direct injection of DNA/dendrimer complexes constitutMOLECULAR THERAPY Vol. 2, No. 6, December 2000 Copyright The American Society of Gene Therapy 1525-0016/00 $35.00
ARTICLE ed with the fifth generation of ethylenediamine (EDA) core dendrimer (G5EDA) increased the efficiency of plasmid gene transfer in vivo and prolonged allograft survival (12). Several approaches for gene transfer into hearts have been investigated in previous studies, such as direct injection, intracoronary perfusion, intrapericardial injection, and systemic gene transfer (13–16). Although direct injection of genes is a simple procedure, this technique, utilizing injection directly into the myocardium in vivo, is limited by transfection of a small number of cells within the needle track and damage or cellular infiltration occurring along the needle track and other sites that come into contact with the needle or injection fluid (13, 17). Intrapericardial gene transfer is also unlikely to achieve widespread cardiac gene expression, and unless vector targeting is made more precise, systemic administration holds little promise for cardiac gene therapy. Intracoronary gene delivery obviates these limitations and has the potential to achieve widespread gene distribution that is limited to the heart, whether the vector is administrated in vivo or ex vivo (14, 18–21). This study investigated the potential utility of dendrimers to enhance gene transfer efficiency in heterotopic vascularized heart transplants by means of perfusion with DNA/dendrimer complexes via the coronary arteries during donor heart harvesting. In addition, we evaluated the ability of agents which increase vascular permeability and therefore interstitial exposure to the vector, including papaverine, VEGF (vascular endothelial growth factor), and serotonin, and prolonged incubation time, to increase gene expression further.
MATERIALS
AND
METHODS
Mice. C57BL/6J (H-2b) (8–10 weeks of age, 17–21 g) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health. Plasmid. pMP6A-β-gal (22) encoding the β-galactosidase (β-Gal) gene under the control of the human cytomegalovirus promoter (HCMVie 1) was a generous gift from Dr. M. Philip (Applied Immune Sciences, Inc., Santa Clara, CA). The plasmids were purified by two cycles of CsCl gradient centrifugation. DNA/dendrimer complexes. Starburst PAMAM dendrimers were synthesized and prepared as previously described (10, 11, 23). The G5EDA was tested in this study. The DNA/dendrimer complexes were prepared by adding 10 µg of DNA plus 130 µg of dendrimer in a total volume of 100 µl in Ringer’s solution, which makes a DNA:dendrimer charge ratio of 1:20, and incubated for at least 10 min at room temperature to allow complex formation. Charge ratios of 1:20 and 1:60 were also prepared using different concentrations of DNA and dendrimers. For some controls, 10 µg of DNA was also mixed with 10 µg of Lipofectamine 2000 (Gibco BRL, Grand Island, NY) in a final volume of 100 µl. Intracoronary gene transfer and abdominal heart transplantation. Heterotopic intraabdominal transplantation was performed as previously described (24). After anesthesia with 4% chloral hydrate (0.1 ml/10 g body wt, ip), the donor heart was arrested by infusion of 0.5 ml of cold lactated Ringer’s solution or cold University of Wisconsin (UW) solution into the inferior vena cava. After ligation of the aortic arch proximal to
MOLECULAR THERAPY Vol. 2, No. 6, December 2000 Copyright The American Society of Gene Therapy
the right subclavian artery, the aortic root was flushed with 0.1 ml of preservation solution to expel intracoronary blood thoroughly, and 0.1 to 0.2 ml of the DNA/dendrimer complexes was then infused into the aortic root with a 30-gauge needle. The heart was removed from the chest and placed in chilled preservative solution. Through a midline abdominal incision, the recipient’s infrarenal abdominal aorta and inferior vena cava were isolated and obstructed both proximally and distally with two microvascular clamps. The donor aorta and pulmonary artery were joined end-to-side to the recipient aorta and vena cava, respectively, using 10-O nylon suture. After the completion of the anastomosis, the proximal and distal clamps were released and the abdomen was closed with a single running suture to all layers. The cold ischemic time (incubation time) was 1 h except in the prolonged incubation groups. The mouse was then warmed for a few hours during recovery from anesthesia and had free access to water and food. The function of the transplanted heart was assessed daily by abdominal palpation. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) staining. All transplanted grafts were functioning when harvested and the hearts were cut into two pieces from apex to base. The graft was snap frozen in liquid nitrogen and embedded in OCT (Miles Scientific, Naperville, IL) and sections were made at 10 µm and collected onto gelatin-coated glass slides. The sections were fixed at room temperature in 0.25% glutaraldehyde in phosphate buffered saline (PBS) for 30 min, rinsed three times in PBS for a total of 30 min, and incubated at 37°C overnight in 0.5 mg/ml X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, 1 mM spermidine, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate in PBS. After staining, the sections were fixed with 4% formaldehyde and counterstained with eosin. Five sections were examined for each mouse. For quantitative analysis of gene expression, the total number of cells positively staining for β-galactosidase was counted per section under magnification (×40), and a mean value was calculated for five sections per graft. The overall mean values were determined for each group. Statistical analysis. Numbers are expressed as means ± standard deviations of the number of stained cells observed. The difference of the amount of gene expression was evaluated with Student’s t test. The level of significance was accepted as P < 0.05.
RESULTS Gene Expression by DNA/Dendrimer Complexes Transferred via Coronary Arteries All donor hearts resumed sinus rhythm several minutes after reperfusion. Total ischemic time of donor hearts was about 60 min, except in the prolonged incubation groups. The technical success rate was greater than 95% in the control groups and lower in some experimental groups, as noted below. The charge ratio of DNA to dendrimer is one of the critical parameters in the optimization of in vitro transfection, and previous studies (10, 11) showed the optimal charge ratio of DNA:dendrimer to be between 1:5 and 1:50 in in vitro experiments. Our previous in vivo study showed that DNA/dendrimer complexes with a charge ratio of 1:50 resulted in highest gene expression after direct injection into nonvascularized cardiac grafts (12). To determine which charge ratio and concentration of DNA and dendrimers can achieve the best gene transfer and expression in the vascularized heart grafts, we tested the complexes at charge ratios of 1:20 and 1:60 with different amounts of DNA and dendrimer. DNA/dendrimer complexes were perfused through graft coronary arteries during heart harvesting,
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FIG. 1. DNA/dendrimer complexes mediate β-Gal gene transfer into murine cardiac grafts. (A) Epicardial gene expression. (B) Endothelial gene expression. (C) Myocardial gene expression. (D) Control.
and the cold ischemic time was kept to 1 h. Grafts were then transplanted to syngeneic recipients who were sacrificed 7 and 14 days after transplantation. Reporter gene expression was noted to be stable over this time frame and waned by 4 to 6 weeks (not shown). The reporter gene expression was determined by X-Gal staining. The grafts perfused with DNA/dendrimer complexes via coronary arteries showed X-Gal staining, with blue-colored cells appearing in epicardial, myocardial, and vascular endothelial cells, but predominantly in myocardial myocytes (Fig. 1). The highest gene expression (190 cells/section) was obtained by perfusion with 200 µl of DNA/dendrimer complexes, which contains 20 µg of DNA and 260 µg of dendrimer (1:20 charge ratio) (Table 1). DNA without dendrimer resulted in no reporter gene expression (Table 1). Mixture of DNA with the commercial lipid reagent Lipofectamine 2000 resulted in far less reporter gene expression than with dendrimer, and no positive staining could be seen with the X-Gal reporter assay (not shown). An interesting finding was that precipitates of particles formed during coronary perfusion with DNA/dendrimer complexes at a charge ratio of
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≥1:60, with subsequent graft thrombosis. We also found that the incidence of graft thrombosis increased with increasing concentrations of dendrimers administered via the coronary arteries, regardless of charge ratio. The results indicate that high concentrations of cationic dendrimer within coronary arteries induced graft thrombosis. The DNA/dendrimer complexes at a charge ratio of 1:20, containing 20 µg of DNA and 260 µg of dendrimers in a total volume of 200 µl, were chosen for intracoronary transfer in further studies.
Serotonin or VEGF Can Enhance Gene Expression Serotonin and VEGF can increase vascular permeability, and previous studies showed that transferred gene expression was increased in rabbit hearts pretreated with serotonin (19). To assess the ability of serotonin or VEGF to augment gene expression with DNA/dendrimer complexes transferred to the heart graft, UW solution containing different concentrations of serotonin or VEGF was infused via the inferior vena cava and coronary arteries in the donor heart in situ. After 5 min, the DNA/den-
MOLECULAR THERAPY Vol. 2, No. 6, December 2000 Copyright The American Society of Gene Therapy
ARTICLE TABLE 1 Analysis of DNA/Dendrimer Transfection Efficiency According to Charge Ratios and Concentration Total transplants
Number of thromboses
100
9
4
112.5 39.4
200
17
4
190.3 51.5
260
100
15
8
152.3 82.7
520
200
2
1
106 53.6
80
1040
200
2
2
—
1:60
10
390
100
2
2
—
—
20
0
200
4
0
0
Group
Charge ratio
1
1:20
2
1:20
3 4
DNA (µg)
Dendrimer
Vol. (µl)
10
130
20
260
1:20
20
1:20
40
5
1:20
6 7
Positive cells/section (meansSD)
* p0.05 vs group 7.
drimer complexes (with or without serotonin or VEGF) were then perfused through coronary arteries and the graft was incubated for an additional 1 h. The results show that 10−5 M serotonin or 1 ng/ml VEGF can enhance gene expression from the DNA/dendrimer complexes transferred to the heart graft (Tables 2 and 3). However, with increasing concentrations or times of exposure to serotonin or VEGF, the incidence of thrombosis of the graft increased. Thus while enhancement of vascular permeability increases gene transfer efficiency, it also worsens graft outcome, likely due to increased graft interstitial edema, promoting vascular thrombosis.
Prolonged Exposure to DNA/Dendrimer Complexes Augments Gene Expression A previous study showed that a 3-h incubation period was required for optimal DNA/dendrimer-mediated gene transfer in in vitro experiments (11). In the current study the interval between the harvest and the reperfusion of the grafts was the incubation period (cold ischemic time), and the incubation period in the studies noted above was 1 h. To examine the possibility that prolonged incubation periods would further enhance DNA/dendrimer incorporation into the graft and gene expression,
we prolonged the exposure time to DNA/dendrimer complexes in lactated Ringer’s solution or UW solution. UW is the most widely used preservation solution for liver, kidney, and pancreas with excellent clinical and experimental preservation data, and UW is considered the current gold standard preservation solution. We compared lactated Ringer’s solution to UW solution for both standard 1-h and prolonged incubation periods. The results show that gene expression was increased with 1.5 h in Ringer’s solution (475 cells/section) or 2 h in UW solution (484 cells/section). However, prolongation to 2 h in Ringer’s solution or 3 h in UW solution resulted in significant graft failure, probably as a result of ischemic injury (Table 4).
Papaverine and Tyloxapol Do Not Increase Gene Transfer with DNA/Dendrimer Complexes Papaverine is a vasodilator, and a previous study showed that papaverine and histamine enhanced gene transfer with a recombinant adeno-associated viral vector in rat hindlimb and heterotopically transplanted hearts (25). To test the enhancement of gene transfer by papaverine for DNA/dendrimer complexes, we pretreated the heart grafts by infusing them with different concen-
TABLE 2 Effects of Serotonin on Gene Expression Total transplants
Number of thromboses
Positive cells/section (meansSD)
10-3 M
4
3
38 13.04
10-4 M
3
3
—
10-3 M
3
2
102 28.64
3 10-5 M
7
2
447.2 34.08*
10-5 M (2 h incubation)
3
2
137 39.18
10-5 M (serotonin in complexes)
4
3
115 39.18
10-6 M (serotonin in complexes)
4
1
290.67 36.15
10-6 M (2 h incubation)
3
3
—
Control (without serotonin)
4
0
160.33 20.11
Group
Note. For the basic experiment, 20 µg DNA plus 260 µg dendrimer (1:20 charge ratio) in 200 µl was infused into donor hearts. Serotonin at the indicated concentration was incorporated into the initial in situ flush solution infused into the inferior vena cava and the aortic root. DNA/dendrimer complexes, without serotonin were then infused and total ischemic time was kept to 1 h. In some groups the incubation (total ischemic) time was increased to 2 h, or the serotonin was also placed into the DNA/dendrimer solution. * p0.05 vs all other groups.
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ARTICLE TABLE 3 Effects of VEGF on Gene Expression Total transplants
Number of thromboses
Positive cells/section (meansSD)
1 ng/ml
3
0
308 49.59*—
25 ng/ml
4
3
290 74.16
1 ng/ml (2 h incubation)
3
3
—
1 ng/ml (VEGF in complexes)
4
2
300 31.27
Control (without VEGF)
4
0
160.33 20.11
Group
Note. As for Table 2, 20 µg DNA plus 260 µg dendrimer in 200 µl was infused into donor hearts, and VEGF was added to the flush solution and/or to the DNA/dendrimer solution. Incubation and ischemic times were 1 or 2 h. * p0.05 vs control.
trations of papaverine (with or without histamine) in UW solution via the inferior vena cava (0.5 ml) and aortic root (0.1 ml). After 5 min, 200 µl of DNA/dendrimer complexes (without papaverine or histamine) was infused via the coronary arteries. This treatment resulted in inferior graft survival, and lower concentrations of papaverine (with or without histamine) that were compatible with graft survival did not augment gene expression (Table 5). Tyloxapol is a detergent component of artificial lung surfactant which in an in vitro study improved gene transfer to, and gene expression in, cell lines and primary cells (26). However, this compound impeded gene expression in an in vivo study of gene transfer to mouse lung (27). We infused DNA/dendrimer complexes in the presence of tyloxapol (25 µg/100 µl) via coronary arteries. The results (not shown) demonstrated no enhancement of gene expression in heart, commensurate with the previous studies of gene delivery to the airway surface in lung in vivo (27).
DISCUSSION Gene therapy for cardiovascular diseases is becoming a clinical reality. Some potential targets for genetic treatment in cardiovascular diseases include coronary disease, heart failure, thrombosis, reendothelialization, and extracellular matrix synthesis. Gene transfer into heart allografts offers the possibility of modulating graft immunogenicity, inhibiting the immune response to alloantigens, and decreasing transplant rejection. A number of studies have shown that a variety of viral vectors (retroviral and adenoviral vectors) and nonviral vectors (plasmids and liposomes) can successfully transfer reporter genes into transplants (1–8). Because of the instability of retroviral particles and the inability of retroviruses to integrate in nonreplicating cells, retroviral vectors will have limited use for direct gene transfer in vivo (27). The adeno-associated virus is gaining attention for its potential use as a human gene therapy vector, but the parvovirus has restricted packaging and takes several weeks to achieve high gene expression, which limits its use in acute intervention protocols, such as transplanta-
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tion. The ability to integrate into the genome of nondividing cells makes lentiviruses particularly attractive in human gene therapy, but the safety of lentiviral vectors is hampered by the potential for replication-competent retrovirus and insertional mutagenesis. Transient, highlevel gene expression can be achieved with replicationdefective adenoviral vectors, but transgene expression is limited over time due to a strong immune response. In addition, innate immune responses to the vector may present an insurmountable safety issue. Conversely, nonviral vectors or plasmid DNA may be used safely, but have limited efficiency. Starburst PAMAM dendrimers, by displaying positive charge densities restricted to the surface of the molecule at physiologic pH (23, 29), have characteristics which allow them to interact simultaneously with both negatively charged phospholipids on cell membranes and DNA. Previous studies showed that DNA/dendrimer complexes were stable for many weeks in solution, unlike lipid mixtures, and capable of mediating highefficiency transfection to a variety of cell lines in vitro. Dendrimers increase and prolong DNA uptake into the cell and nucleus through energy-dependent endocytosis (10). Our recent study demonstrated that Starburst dendrimers could be used to mediate efficient in vivo transfer of plasmid DNA into nonvascularized murine cardiac grafts, increasing the efficiency of gene transfer and extending gene expression (12). Several gene transfer approaches to hearts have been TABLE 4 Analysis of Incubation Times and Preservative Solutions Group
Total transplants
Number of thromboses
Positive cells/section (meansSD)
1 h (UW)
20
4
165.533 24.11
2 h (UW)
10
2
484 26.94
2 h (UW)
7
7
—
1 h (LR)
4
0
160.3 51.5
1.5 h (LR)
10
2
475 58.9
2 h (LR)
4
3
90 26.4
Note. 20 µg DNA plus 260 µg dendrimer (1:20 charge ratio) in 200 µl infused into heart grafts.
MOLECULAR THERAPY Vol. 2, No. 6, December 2000 Copyright The American Society of Gene Therapy
ARTICLE TABLE 5 Effects of Papaverine Total transplants
Number of thromboses
Positive cells/section (meansSD)
0.8 mM papaverine
5
5
—
0.5 mM papaverine
4
4
—
0.1 mM papaverine
3
0
10
0.1 mM papaverine
6
6
—
4
0
65.1 13.18
Group
(in 10 mM histamine)
0.1 mM papaverine (in 3 mM histamine)
Note. 20 µg DNA plus 260 µg dendrimer (1:20 charge ratio) in 200 µl infused into heart grafts.
investigated in previous studies. When systemic administration of recombinant replication-defective adenoviral vector was used, only minor myocardial uptake was observed (16). A recent study showed that the myocardium was transfected after an adenoviral vector was injected into the pericardial sac (15). Intramuscular gene transfer represents an alternative method to target the myocardium (28). In vivo, direct myocardial gene transfer through a needle has been investigated in the rat, rabbit, pig, and dog (13, 30–33). Although direct injection of genes into the myocardium is a simple procedure, this technique is limited by transfection of a small number of cells around the injection site and gene expression is temporally limited as well, with expression peaking within several weeks and declining rapidly thereafter (30, 31). Postinjection hearts showed acute inflammatory response along the track of the needle, and fibrosis along the needle track also was observed (30). Gene transfer through coronary arteries provides access to the entire myocardium. A number of recent studies have shown that gene transfer and gene expression are more efficient and widespread than the direct injection approach (19, 21, 34–36). In this study, we used DNA/dendrimer complexes to infuse the aortic root. The results clearly demonstrate that the hearts were transfected with β-galactosidase cDNA when DNA/dendrimer complexes were administered by coronary infusion during the donor heart harvest. The DNA was transferred to cardiac myocytes both in the epicardium and in the endocardium of the heart and to the vascular endothelium. Gene expression was optimized by infusion of 200 µl of DNA/dendrimer complexes containing 20 µg of DNA and 260 µg of dendrimers in a charge ratio of 1:20. A number of maneuvers were investigated to enhance gene expression further. Previous studies demonstrated the ability of serotonin to increase microvascular leak (37) by acting at the level of the postcapillary venules to increase permeability through endothelial contraction and disruption of tight junctions. Our results showed enhancement of β-galactosidase gene transfer and expression after pretreatment with serotonin, and the result is consistent with that previous study (19). Our MOLECULAR THERAPY Vol. 2, No. 6, December 2000 Copyright The American Society of Gene Therapy
study also demonstrated that prolongation of exposure to DNA/dendrimer complexes (2 h) further enhanced gene transfer and expression in the cardiac transplants. Presumably, increased contact time promotes binding and internalization of the complex. However, more prolonged incubation (3 h) might not be feasible for heart transplantation as this organ is extremely sensitive to ischemic damage. In this study, we could not confirm that papaverine plus histamine or tyloxapol could enhance gene transfer and gene expression with DNA/dendrimer complexes in cardiac allografts, although other studies showed that each could increase gene expression in different models (25, 26). This likely represents idiosyncrasies of species, strains, and in vivo experimentation. In conclusion, this study demonstrates that the cardiomyocytes of heart grafts express an exogenous gene after DNA/dendrimer complex gene transfer via the coronary arteries under hypothermic preservation conditions. Serotonin and prolonged incubation time enhance gene transfer and gene expression in the cardiac transplants. ACKNOWLEDGMENTS This work was supported by Baxter Extramural Grant Program and NIH Grant AI42840 (both to J.S.B.). We thank Dr. M. Philip for the pMP6A-β-gal plasmid.
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MOLECULAR THERAPY Vol. 2, No. 6, December 2000 Copyright The American Society of Gene Therapy
