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Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods

MIKKO O. HILTUNEN^*,1, MIKKO P. TURUNEN^*,1, ANNA-MARI TURUNEN^*, TUOMAS T. RISSANEN^*, MARJA LAITINEN^*,, VELI-MATTI KOSMA^§ and SEPPO YLÄ-HERTTUALA^*,,2

^* A. I. Virtanen Institute,
Department of Medicine,
Gene Therapy Unit and
^§ Department of Pathology and Forensic medicine, University of Kuopio, Kuopio, Finland

²Correspondence: Department of Molecular Medicine, A. I. Virtanen Institute, University of Kuopio, FIN-70211 Kuopio, Finland. E-mail: Seppo.Ylaherttuala{at}uku.fi

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expressionof transgene other than in the target tissue may cause side effects and safety problems in gene therapy. We analyzed biodistribution of transgene expression after intravascular and periadventitial gene delivery methods using the first generation nuclear-targeted lacZ adenovirus. RT-PCR and X-Gal stainings were used to study transgene expression 14 days after the gene transfer. After intravascular catheter-mediated gene transfer to rabbit aorta mimicking angioplasty procedure, the target vessel showed 1.1% ± 0.5 gene transfer efficiency. Other tissues showed varying lacZ gene expression indicating a systemic leakage of the vector with the highest transfection efficiency in hepatocytes (0.7% ± 0.5). X-Gal staining of blood cells 24 h after the intravascular gene transfer indicated that a significant portion (1.8% ± 0.8) of circulating monocytes was transfected. X-Gal-positive cells were also found in testis. After periadventitial gene transfer using a closed silicon capsule placed around the artery, 0.1% ± 0.1 lacZ-positive cells were detected in the artery wall. Positive cells were also found in the liver and testis (<0.01%), indicating that the virus escapes even from the periadventitial space, although less extensively than during the intravascular application. We conclude that catheter-mediated intravascular and, to a lesser extent, periadventitial gene transfer lead to leakage of adenovirus to systemic circulation, followed by expression of the transgene in several tissues. Possible consequences of the ectopic expression of the transgene should be evaluated in gene therapy trials even if local gene delivery methods are used.—Hiltunen, M. O., Turunen, M. P., Turunen, A.-M., Rissanen, T. T., Laitinen, M., Kosma, V.-M., Ylä-Herttuala, S. Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods.

Key Words: adenovirus • biodistribution • vascular gene therapy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
DURING THE LAST few years, gene transfer techniques have rapidly emerged as an alternative approach for treating various genetic and acquired diseases (1) . The use of plasmids and viral gene transfer systems has demonstrated that genes can be transferred to various cell types in vitro and in vivo with variable efficiency. A direct therapeutic effect on the pathological process in the target tissue can be achieved with local gene transfer. We and others have demonstrated the efficiency of gene transfer to vascular wall and malignant glioma with various gene transfer vectors (plasmid/liposomes, plasmid/polymer complexes, second generation pseudotyped retroviruses, and adenoviruses) in experimental animals and in humans (2 3 4 5) . However, the systemic expression of the transgene in nontarget tissues has not been systematically evaluated although it has been recognized that ectopic transgene expression is a possible drawback of gene therapy (6) .

Local intravascular adenovirus-mediated gene transfer to vessel wall can be performed using angioplasty and several other types of catheters leading to the transfection of endothelial cells and smooth muscle cells (6 7 8) . In the local periadventitial gene transfer to arterial wall applicable during vascular surgery, the transfected cells are usually fibroblasts and smooth muscle cells (5) . After a systemic tail vein injection of adenovirus in mice, it has been demonstrated by sensitive nested polymerase chain reaction (PCR) methods that virus DNA can be found in various tissues, such as liver, lung, kidney, and testis (9) . However, despite the large number of clinical gene therapy trials, there is a shortage of basic in vivo information regarding the spread of transgenes to nontarget tissues after local gene delivery methods are used. Accordingly, safety aspects and biodistribution of the vectors need to be better understood before safe tools for clinical treatments can be developed.

The purpose of this study was to evaluate tissue distribution of adenovirus vector and transgene expression after intravascular and periadventitial gene transfer in rabbits. It was found that systemic leakage of the virus and expression in nontarget tissues occurred with both gene transfer methods, although there was much less leakage with periadventitial than with intravascular delivery.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Adenovirus vector
Ad5 serotype (E1, partial E3-deletion) was used in the current study (10) . Adenovirus was cloned using homologous recombination of pAdenogal plasmid (11) . Replication-deficient E1-E3 deleted adenoviruses were produced in 293 cells. After homologous recombination and isolation of virus-containing lysates, three rounds of plaque purification were used to isolate a pure clone of Ad5 expressing the lacZ transgene. Virus was purified through two rounds of CsCl gradient ultracentrifugation, followed by dialysis and storage in buffered 20% glycerol at -70°C (4 , 11) . Human clinical grade lacZ adenoviruses were produced under GMP conditions (MediGene Oy, Kuopio, Finland) (4 , 11) . Final lot release tests for the adenoviruses included titer assay on 293 cells, Southern blotting, E1/E2 selective PCR analysis, transient transfection analysis in vitro and cytopathic effect assay on A549 cells (12) . All viral lots were also analyzed for the absence of microbiological contaminants (2 wk sterility), mycoplasma (PCR assay), and lipopolysaccharide (limulus assay; endotoxin level <10 EU/ml) (12) . The lacZ gene construct has a CMV promoter as well as a nuclear targeted signal that directs the ß-galactosidase activity into the nucleus of the target cell (4 , 11) . lacZ adenovirus product contained 110–115 virus particles/plaque forming unit.

Animal models
Gene transfer was done to New Zealand white rabbit abdominal aorta using a 3.0 F channeled balloon local drug delivery catheter (13) (Boston Scientific Corp., Maple Grove, Mass.). Under fluoroscopical control, the balloon catheter was positioned caudal to the left renal artery in a segment free of side branches via a 5 F percutaneous introducer sheath (Arrow International, Reading, Pa.) in the right carotid artery and inflated at 6 ATM with a mixture of contrast media and saline. 1.15 x 10¹⁰ pfu of adenovirus was used in the final volume of 2 ml at 6 ATM pressure for 10 min. Animals were killed 14 days (n=3) after the gene transfer.

For studying periadventitial gene transfer, we used a local, closed periadventitial gene delivery method (2 , 5) . Through a midline neck incision, the common carotid arteries were exposed between sternohyoid and sternocleidomastoid muscles. The arteries were gently prepared from surrounding tissues and silastic collars were installed around the arteries. Gene transfer was done immediately after the collar installation using 1.15 x 10¹⁰ pfu adenovirus. Animals were killed 14 days (n=3) after the operation. Three equally treated rabbits who received adenovirus that did not contain lacZ were used as controls for the intravascular delivery and three rabbits were used for the perivascular delivery. All studies were approved by the Experimental Animal Committee of the University of Kuopio, Finland.

X-Gal staining of tissues and blood cells
Tissue samples for ß-galactosidase assay were fixed in 4% paraformaldehyde/0.15 M sodium-phosphate buffer for 10 min and rinsed with 0.15 M sodium-phosphate buffer (pH 7.2) for 2 h. Arteries were then embedded in OCT compound (Miles, Elkhart, Ind.) and stored at -70°C. The X-Gal reagent (100 mg/ml) was prepared by dissolving 1 g X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) (MBI Fermentas, Germany) to 10 ml dimethyl formamide (14) . The X-Gal reagent was diluted dropwise 1:100 with X-Gal solution [5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 2 mM MgCl₂] by slowly mixing to avoid precipitation. Frozen sections were stained in the dark at +37°C for 8 h. Sections were rinsed with phosphate-buffered saline, dehydrated with rising ethanol concentrations, and counterstained with Mayer’s Carmalum (5) . Micrographs were taken by Olympus AX 70 microscope (Olympus Optical, Tokyo, Japan). The transfection efficiency was measured by X-Gal staining of 10 randomly selected sections from each tissue sample and calculated using the equation: transfection efficiency (%) = (X-Gal-positive cells/total number of cells in a section) x 100% (4 , 11) . Control samples were taken from rabbits treated with an adenovirus that did not contain lacZ. The same samples were also used as negative controls for reverse transcriptase (RT) -PCR (see below).

For the X-Gal staining of blood cells, the mononuclear fraction was isolated using NycoPrep (Nycomed Pharma AS, Oslo, Norway). Mononuclear cells were pipetted on fibronectin coated slides (Becton Dickinson Labware, Bedford, Mass.) and left to attach for 12 h in the presence of cell culture medium (DMEM, 10% fetal bovine serum; Gibco BRL, Rockville, Md.). The medium was removed and cells were incubated in X-Gal solution for 7 h. The X-Gal staining solution was discarded; cells were counterstained with Mayer’s Carmalum and processed for light microscopy.

RT-PCR
Total RNA was extracted using Trizol Reagent (Gibco BRL) according to the manufacturer’s instructions and cDNA synthesis was performed with 4 µg of total RNA using random hexamer primers (Promega, Madison, Wis.). The reaction was carried out in a total volume of 35 µl for 1 h at +37°C and then heated to +95°C to denature the enzyme. Nested PCR was used to amplify lacZ mRNA from tissues. The primers for the first PCR were designed to distinguish between endogenous lacZ and transduced lacZ by selecting the 5' primer (5'-TTGGAGGCCTAGGCTTTTGC-3') from CMV promoter and the 3' primer (5'-ATACTGTCGTCGTCCCCTCA-3') from lacZ gene. The first PCR reaction was performed with 10 µl of synthesized cDNA of each sample. Each reaction mixture contained 20 pmol of each primer, 200 µM of each deoxynucleotide triphosphate (Promega), 1.5 mM MgCl₂, 2% deionized formamide, and 1 U of recombinant Taq DNA polymerase (MBI Fermentas) in addition to 1x PCR reaction buffer (MBI Fermentas) containing 10 mM Tris-HCl, pH 8.8, 50 mM NaCl, and 0.08% Nonidet P 40. The first PCR consisted of 30 cycles of +94°C 45 s, +58°C for 45 s, and +72°C for 50 s. The second PCR used 32 cycles of +94°C for 1 min, +58°C for 1 min, and +72°C for 90 s. The nested primers for the second PCR were 5'-GGTAGAAGACCCCAAGGACTTT-3' and 5'-CGCCATTCGCCATTCAG-3'.

Clinical chemistry
Serum samples were collected at baseline and 2, 7, and 14 days after the gene transfer. C-reactive protein (CRP), alkaline phosphatase, alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatinine (crea) were monitored from serum samples using routine clinical chemistry assays at Kuopio University Hospital Central Laboratory. Anti-adenovirus antibodies (adv-ab) were measured using a standard complement fixation assay (BioWhittaker, Walkersville, Md.) (15) .

	RESULTS

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Representative examples of the X-Gal stainings of major tissues after intravascular catheter-mediated and local periadventitial collar-mediated gene transfers are presented in Fig. 1 and Fig. 2 , respectively. A 14 day time point was selected for the studies because it was assumed that if the transgene expression in ectopic tissues is present after such a long period, the likelihood of unwanted side effects may truly be a matter of concern. The percentages of transfected cells in various tissues after the gene transfer are listed in Table 1 and RT-PCR results are shown in Fig. 3 and Table 1 . Gene transfer to rabbit aorta using channeled balloon catheter resulted in 1.1% ± 0.5 gene transfer efficiency in the target tissue 14 days after the gene transfer (Table 1) . The lacZ marker gene expression was localized mostly in the intima. Some expression was also found in the media. Periadventitial gene transfer resulted in 0.1% ± 0.1 gene transfer efficiency. Positive cells were localized in the adventitia. The majority of transfected cells in intima and media were smooth muscle cells as determined by -actin-specific immunostaining (data not shown). Positive cells in adventitia were mainly fibroblasts.

View larger version (124K): [in this window] [in a new window]   Figure 1. Nuclear targeted lacZ expression in various tissues 14 days after intravascular catheter-mediated adenovirus (1.15 x 1010 pfu) gene transfer. a) Artery wall at the site of gene transfer. b) Control artery. c) Psoas major muscle. d) Testicular tissue. e) Epididymis. f) Sperm. g) Liver. h) Heart muscle. i) Lung. j) Cerebra. k) Cerebellum. l) Spleen (area showing maximal staining in red pulp). m) Kidney. n) Bone marrow. o) Isolated blood monocytes. X-Gal staining. Bar in each figure corresponds to 50 µm. Arrows indicate positive cells.

View larger version (135K): [in this window] [in a new window]   Figure 2. Nuclear targeted lacZ expression in various tissues 14 days after local periadventitial collar-mediated adenovirus (1.15 x 1010 pfu) gene transfer. a) Artery wall at the site of gene transfer. b) Control artery. c) Cervical lymph node. d) Sternocleidomastoid muscle. e) Cerebra. f) Cerebellum. g) Heart muscle. h) Lung. i) Spleen (area showing maximal staining in red pulp). j) Kidney. k) Testicular tissue. l) Epididymis. m) Sperm. n) Liver. o) Bone marrow X-Gal staining. Bar in each figure corresponds to 50 µm. Arrows indicate positive cells.

View larger version (32K): [in this window] [in a new window]   Figure 3. RT-PCR of tissues 14 days after gene transfer. Positive PCR product size is 218 bp. Tissues after local intravascular gene transfer are in the upper panel (I) and tissues after periadventitial gene transfer are in the lower panel (II). From the left: molecular weight marker (MW), artery at the site of the gene transfer (A), control artery (B), bone marrow (C), white blood cells (D), skeletal muscle (E), testis (F), epididymis (G), sperm (H), liver (I), heart (J), lung (K), brain (L), cerebellum (M), spleen (N), kidney (O), and molecular weight marker (MW). See Materials and Methods for details of the nested RT-PCR analysis.

The highest ectopic marker gene expression was found in the liver, where 0.7% ± 0.5 of hepatocytes were transfected after intravascular gene transfer. Positive cells were also found in the liver after periadventitial gene transfer, but to a lesser extent (<0.01%, Table 1 ).

Marker gene expression was found both in testis and epididymis after intravascular gene transfer. After periadventitial gene transfer, positive cells were found in testis, but not in epididymis (Table 1) . No positive signals were found inside the tubules or in germ cells by using either of the gene transfer methods.

X-Gal-positive alveolar macrophages and bronchiolar epithelial cells were found in lung tissue after the intravascular gene transfer. In the spleen positive cells were seen mostly in the red pulp, but also to a lesser extent in the white pulp. In the kidney, the positive cells were localized within the vasculature. After intravascular gene transfer no transfected cells were found in skeletal muscles near the gene transfer area with the X-Gal staining method, but according to RT-PCR, psoas major muscle expressed exogenous lacZ mRNA (Fig. 3 .). After periadventitial gene transfer, positive cells were found in skeletal muscle. Spleen, kidneys, and lungs stained negative after periadventitial gene transfer. Heart muscle was negative in X-Gal stainings after both gene transfer methods, but positive in RT-PCR. Positive cells were found in the cervical lymph nodes after periadventitial gene transfer. Cerebra and cerebellum were negative in X-Gal stainings and RT-PCR for every animal in both groups.

According to X-Gal staining, 1.8% ± 0.8 of white blood cells were transfected with adenovirus 24 h after intravascular gene transfer, but no such transfection was seen after periadventitial gene transfer. The majority of the transfected cells were monocytes. No ß-galactosidase was detected in any sections or blood samples from control animals in histology or in RT-PCR.

Clinical chemistry showed transient elevations in CRP, ALT, and LDH and an increase in anti-adenovirus antibodies after both gene transfer methods (Table 2 ).

	DISCUSSION

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It has recently been realized that severe safety problems may be associated with adenovirus-mediated gene therapy (16) and that some of the side effects may be caused be systemic spread and expression of the adenoviruses in ectopic tissues. It is surprising that very little information is available about the systemic distribution of adenoviruses after local gene transfer, since most investigators have apparently thought that vectors administered locally will stay at the site of transfection. In this study we addressed this issue using intravascular and periadventitial delivery routes for vascular gene transfer. The former route of administration mimics percutaneous angioplasty procedure. The latter method can be used during vascular surgery by placing a hollow, closed silastic or biodegradable collar or capsule around the artery (2 , 5) . Collar model has been used to study effects of gene transfer and other therapeutic approaches on neointima formation (3 , 17 , 18) . When designing these experiments we hypothesized that, in the angioplasty setup, a systemic escape of the adenovirus is likely but should not occur when using the perivascular closed gene transfer capsule. However, our results demonstrate that 1) both routes of administration lead to systemic spread of viruses; 2) clinical chemistry suggests that the systemic viral load may be sufficient to cause transient hepatic dysfunction; and 3) humoral immune response against adenovirus was evident after both gene delivery methods.

Hepatocyte was the most prominent nontarget cell type transfected with the intravascular gene transfer. As reported previously, this can produce severe clinical problems when, for example, suicide gene therapy is used (19) . Hepatocytes were also transfected after periadventitial gene transfer. It appears that adenoviruses can escape from the perivascular space to systemic circulation via vasa vasorum or through lymphatics. Since no positive cells were seen in the media or intima after periadventitial gene transfer, it is unlikely that adenoviruses were able to penetrate directly into vascular lumen through the vessel wall. Since cervical lymph nodes close to the gene transfer region stained positive for ß-galactosidase activity, it is most likely that lymphatics were involved in the systemic leakage of adenoviruses.

In the spleen, X-Gal-positive cells were seen almost exclusively in the red pulp, and transfection was evident after both gene transfer methods. This observation may be related to the fact that red pulp has loose fenestrated endothelium. Whether transgene expression in spleen contributes to immunological changes seen after adenovirus infection remains to be studied. In skeletal muscle, the majority of X-Gal-positive cells were myocytes.

It is especially important that transfected monocytes were detected in circulating blood 24 h after the intravascular gene transfer. This did not occur after periadventitial gene transfer. Whether monocytes were transfected directly in the blood or in the extravascular space, such as the spleen, remains unknown. The potential significance of this finding for both biosafety aspects and possible therapeutic applications needs to be further studied. Since macrophages normally circulate through tissues, the transduction of blood-borne macrophages may establish transgene expression in many organs. This accumulation may be even more pronounced at sites of adenovirus-mediated tissue damage. On the other hand, it may also be possible to use ex vivo transfected monocytes for systemic delivery of transgenes for therapeutic or diagnostic applications.

We show that a positive RT-PCR signal can be found from the testis and epididymis and that X-Gal-positive cells are located near the vasculature in the testicular tissue. Both gene transfer methods produced X-Gal-positive cells in the testis, but only intravascular gene transfer transfected cells in the epididymis. It has been shown that no inadvertent germ line transmission or demonstrable negative effects were detected after systemic tail vain administration of adenovirus vector in male and female mice (9) . However, because transfection occurs very close to maturating sperm, the possibility of inadvertent germ line gene transfer cannot be fully excluded if an arterial route of transfection is used. No X-Gal-positive sperm was detected in histology; instead, a positive RT-PCR signal was found. In separate experiments, we transduced sperm samples in vitro using adenovirus encoding ß-galactosidase, but detected no X-Gal-positive sperm cells 20 h after the transfection (data not shown). This suggests that sperm cells may not be easily transfected or are not capable of expressing ß-galactosidase. Instead, all sperm samples were contaminated to some extent with blood cells which may give rise to the positive signal in RT-PCR.

It should be recognized that our results may not directly apply to humans since adenovirus is not normally a rabbit pathogen and intra-aortic delivery is not an option for clinical gene therapy. However, intra-aortic delivery is reminiscent of intracoronary application, and adenoviral infection occurs in rabbit tissues in a similar manner as in humans. Periadventitial delivery has been used in human clinical trials (for a review, see ref 1 ) and our results should be applicable to humans. Our data also establish that transduction efficiencies in the vasculature are remarkably low (1% using intravascular route and 0.1% using perivascular route). Thus, it can be concluded that neither delivery method is yet optimal for vascular gene therapy (i.e., good transgene expression in the target tissue and low expression in nontarget tissues). Improvement in the local transfection efficiency could be achieved by better device design or by increasing the viral titer. However, the latter alternative cannot be suggested for the first generation adenoviruses, since increasing the dose would probably only lead to a more widespread expression in ectopic tissues. Note that our results apply only for the first generation adenoviruses and delivery techniques described. Thus, for other applications, such as intratumoral injections (4) , biodistribution may be different. Also, development of modified viruses engineered to express cell-specific tropism by introduction of novel ligands onto the viral capsid or the use of tissue-specific and regulated promoters may show more favorable delivery and expression patterns.

We conclude that catheter-mediated intravascular and, to a lesser extent, periadventitial gene transfer lead to leakage of adenovirus to systemic circulation and that the transgene is expressed in several ectopic tissues. Therefore, we recommend that in each gene therapy trial the consequences of transgene expression in nontarget tissues should always be evaluated on the basis of the function of the transgene and the specific organ. Second, the safety of gene therapy can be improved by using tissue-specific and regulated promoters and more specific, targeted in vivo gene delivery methods.

	ACKNOWLEDGMENTS

 
This study was supported by grants from Finnish Academy, The Finnish Cultural Foundation of Northern Savo, and Kuopio University Hospital (EVO grants 5130). The authors wish to thank Ms. Mervi Nieminen, Ms. Eila Pelkonen, and Ms. Maiju Jääskeläinen for technical assistance, Ms. Marja Poikolainen for preparing the manuscript, and Boston Scientific for providing the gene transfer catheters.

	FOOTNOTES

 
¹ These authors contributed equally to this work.

Received for publication March 27, 2000. Revision received May 15, 2000.

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