Nonviral gene transfer into fetal mouse livers (a comparison ... - Nature

Gene Therapy (2003) 10, 810–817 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt

RESEARCH ARTICLE

Nonviral gene transfer into fetal mouse livers (a comparison between the cationic polymer PEI and naked DNA) H Gharwan1, L Wightman2, R Kircheis2, E Wagner3 and K Zatloukal1 1

Institute of Pathology, University of Graz, Auenbruggerplatz, Graz, Styria, Austria; 2Boehringer Ingelheim R&D Vienna, Dr Boehringer-Gasse, Vienna, Austria; and 3Pharmaceutical Biology – Biotechnology, Ludwig-Maximilians-University Munich, Munich, Germany

We investigated the efficacy and safety of the cationic polymer polyethylenimine (PEI) as a potential tool for intrauterine gene delivery into livers of fetal mice in the last trimester of pregnancy (E17.5). Using luciferase as a reporter gene, transferrin-conjugated and ligand-free PEI/ DNA complexes (containing 3 mg DNA) with varying PEInitrogen/DNA-phosphate (N/P) ratios and different PEI forms, branched (800, 25 kDa) and linear (22 kDa), were compared with naked DNA. Transgene expression was measured 48 h after administration of PEI/DNA complexes or naked DNA. Highest luciferase activity (9.8 103 relative light units (RLU)/mg of tissue protein) was observed with ligand-free PEI22/DNA mixtures at N/P 6.0. In addition, this formulation was associated with very low toxicity as compared to the other PEI/DNA-injected groups. Using

b-galactosidase as a reporter gene, transfection of single, but also small, clusters of cells was demonstrated throughout the liver. Injection of 3 mg naked DNA resulted in an 11-fold lower transgene expression value (0.9 103 RLU/mg of tissue protein) as compared to PEI22/DNA complexes. However, the administration of higher concentrated naked DNA (9 mg) into fetal livers yielded expression levels of 3.2 104 RLU/mg of tissue protein, a more than three-fold increase compared to PEI22/DNA complexes. Furthermore, the gene transfer efficacy of concentrated naked DNA was approximately 40 times higher in fetuses than in adults (0.8 103 RLU/mg of tissue protein), indicating that fetal tissue is especially amenable to the uptake and expression of naked DNA. Gene Therapy (2003) 10, 810–817. doi:10.1038/sj.gt.3301954

Keywords: polyethylenimine; naked DNA; fetus; in utero gene transfer; germ cells

Introduction Prenatal gene delivery could provide the basis for new therapeutic strategies for a variety of genetic and nongenetic diseases. Apart from inherited abnormalities with the requirement for long-term expression of the delivered gene, several noninherited pre- and perinatal conditions, where short-term gene expression is regarded as sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal gene therapy could be the deficiency of the surfactant protein B inducing perinatal respiratory distress, growth factor deficiencies leading to hypoxic injuries of the fetal/neonatal brain,1 or the prevention of a premature closure of the Ductus Arteriosus Botalli.2 Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention very early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult Correspondence: Dr K Zatloukal, Institute of Pathology, Karl-FranzensUniversity of Graz, Auenbruggerplatz 25, A-8036 Graz, Austria Received 11 February 2002; accepted 5 November 2002

organs, which could allow a more efficient gene transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual’s immune system is not completely matured. Therefore, supplementation of a nonfunctional gene by a correct gene version does not cause immune sensitization to the vector and the transgene product, a problem that could arise if gene transfer is performed in adults with a fully functional immune system.3 In the last years, gene transfer into fetuses of various animals has primarily utilized viral vectors.4–7 However, since viral vectors often exhibit side-effects,8,9 safety concerns have led to the investigation of nonviral vectors as alternative approaches.10,11 In the present study, the cationic polymer polyethylenimine (PEI) was used for intrauterine gene transfer. PEI’s ability to condense and deliver DNA to cells has been extensively characterized in vivo,12–15 but the applicability of this substance for prenatal gene transfer has not been addressed to date. A target organ that is particularly interesting for in utero gene delivery is the fetal liver. In the developing fetus, hepatocytes are proliferating at a high rate, with the vasculature being more leaky than in adult animals. These conditions were previously described as advantageous for gene transfer with PEI.14 Furthermore, as mitotically active

Nonviral gene transfer into fetal mouse livers H Gharwan et al

cells overexpress the transferrin receptor,16,17 the addition of transferrin into PEI/DNA complexes might increase their uptake into the target cell via receptormediated endocytosis.14 In this work critical parameters of different PEI/DNA complexes formed under varying conditions were investigated for effective intrauterine gene delivery. Furthermore, it was evaluated whether transgene expression after local injection of the complexes into the fetal liver occurs in tissues other than the target organ.

Results Electron microscopy of different PEI/DNA complexes and evaluation of transfection efficacies in vitro To assess the impact of size and structure of different PEI/DNA complexes on prenatal gene delivery, complexes of 3 mg plasmid DNA with varying forms of PEI (branched and linear) were generated under various conditions (ligand-free or transferrin-conjugated) in saltcontaining (0.5 HBS) or salt-free (HBG) buffers at different N/P ratios (4.8, 6.0 or 7.2). Prior to the in utero administration into fetal mouse livers, the particles were investigated by electron microscopy (Figure 1a–c). Using PEI25/DNA complexes formed under salt conditions at N/P 6.0, small spheres and great aggregates of irregular shapes and different sizes were observed depending on the concentration of transferrin. The less the transferrin added, the less the aggregation was observed, but distinct particles were formed. With PEI22/DNA, complexes formed in salt-containing solutions led to the formation of large aggregates, whereas under salt-free conditions small particles were seen. The functionality of the various complexes was tested in parallel in vitro using b-galactosidase as the reporter

gene. Transfections of 3T3-fibroblasts demonstrated that transgene expression was more efficient with transferrin/PEI800/DNA complexes generated in 0.5 HBS than with PEI25, and was more efficient with PEI25 than with PEI22 (Figure 1d-f).

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Evaluation of adverse effects on pregnancy after intrauterine application of PEI/DNA complexes The influence of the physicochemical properties of different PEI/DNA complexes on the survival of fetal mice was investigated by administration of the particles into fetal mouse livers at day 17.5 of gestation (E17.5). The time of birth of the injected fetuses was predicted to be 48 h later. With certain PEI/DNA complexes, low fetal survival rates were observed (Table 1). Particularly low survival was associated with Tf-PEI800/DNA at N/P 7.2 generated in 0.5 HBS, where no fetus survived the gene transfer procedure (no survivor out of 19 treated fetuses; 0/19). However, at N/P 6.0 and under the same ionic conditions, survival rate increased to 67% (18/27) and at N/P 4.8 even further to 83% (20/24). With PEI25/DNA generated in 0.5 HBS at N/P 6.0, coupling of transferrin to the complexes was important for fetal survival showing a linear correlation between fetal survival and amount of ligand added. In this group, best survival rates (94%) were obtained when transferrin was incorporated at high densities (ratio 1 : 4) into the complexes, whereas survival was lowest (24%) with ligand-free PEI25. With ligand-free PEI22/DNA generated in 0.5 HBS, even up to 90% (27/30) of the injected fetuses survived. Addition of high concentrations of transferrin (ratio 1 : 5) to PEI22-based complexes resulted in 38% (10/26) or 25% (7/28) survival in salt-containing or salt-free environment,

Figure 1 Electron microscopy of different PEI/DNA complexes and evaluation of transfection efficacies in vitro. (a–c) Electron microscopy of transferrinconjugated PEI/DNA complexes (ratio 1 : 5) generated in 0.5 HBS at N/P 6.0. (a) Tf-PEI/PEI800, (b) Tf-PEI/PEI25, (c) Tf-PEI/PEI22. The bar represents 500 nm (a–c). (d–f) LacZ staining of 3T3 cells transfected with the complexes shown in (a–c). (d) Tf-PEI/PEI800, (e) Tf-PEI/PEI25, (f) Tf-PEI/ PEI22. Gene Therapy


812 Table 1

Survival rate of mice after in utero gene transfer

Injection solution

N/P-ratio

Survived fetuses/ injected fetuses

Tf-PEI800/DNA Tf-PEI800/DNA Tf-PEI800/DNA

4.8 6.0 7.2

20/24 (83%) 18/27 (67%) 0/19 (0%)

6.0 6.0 6.0 6.0 6.0

16/17 (94%) 21/27 (78%) 13/33 (39%) 8/30 (27%) 8/33 (24%)

6.0 6.0

27/30 (90%) 10/26 (38%)

Tf-PEI/PEI25/DNA Tf-PEI/PEI25/DNA Tf-PEI/PEI25/DNA Tf-PEI/PEI25/DNA PEI25/DNA

1:4 1:5 1:10 1:20

PEI22/DNA Tf-PEI/PEI22/DNA 1:5 0.5 HBS (as control) HBG (as control)

22/22 (100%) 24/24 (100%)

3 mg naked DNA 6 mg naked DNA 9 mg naked DNA

15/16 (94%) 17/19 (89%) 12/14 (86%)

Viable born mice 48 h after in utero administration (at E17.5) of various PEI/DNA complexes (generated in 0.5 HBS with various transferrin (Tf) contents and N/P ratios), different amounts of naked DNA, and 0.5 HBS or HBS as controls.

respectively, thus having a negative effect on fetal survival. High survival rates of 94% (15/16) were seen with low-dose (3 mg) naked DNA. Increasing the concentration of in utero administered naked DNA to 6 or 9 mg slightly reduced survival rates to 89% (17/19) and 86% (12/14), respectively. Fetuses injected with 0.5 HBS or HBG served as controls and were all delivered with no fetal mortality (22/22 or 24/24).

Gene expression of PEI/DNA complexes after injections into fetal, neonatal or adult livers Using PEI25/DNA, transgene expression values up to 8.8 10371 103. relative light units (RLU)/mg of tissue protein (106 RLU correspond to 2 ng luciferase) were achieved in fetal livers, when the complexes were coupled to transferrin (ratio 1 : 5) and generated in the presence of salt at N/P 6.0 (Figure 2a, lane 3), whereas the administration of ligand-free PEI25/DNA led to only 1.5 10370.7 103 RLU/mg of tissue protein under the same ionic conditions (Figure 2a, lane 1). In contrast to PEI25, no reporter gene expression was detected using transferrin-conjugated PEI800/DNA complexes in pre- or in postnatally injected mice, independent of the conditions used (N/P 4.8, 6.0 or 7.2). Furthermore, no

Figure 2 Luciferase expression in mouse livers and lungs after administration of various PEI/DNA complexes. Luciferase expression (RLU) per milligram tissue protein in livers (a, c and e) and lungs (b, d and f) of mice injected at different ages. Samples with no detectable luciferase expression are indicated as n.d. PEI/DNA complexes (ligand-free or transferrin-conjugated (Tf-PEI) at ratios 1 : 4, 1 : 5, 1 : 10 or 1 : 20) were generated with a total of 3 mg DNA in 0.5 HBS at N/P 6.0 and compared with naked DNA. Transgene expression was measured 48 h after gene transfer by luciferase assay. (1) ligand-free PEI25/DNA, (2) Tf-PEI/PEI25/DNA (1 : 4), (3) Tf-PEI/PEI25/DNA (1 : 5), (4) Tf-PEI/PEI25/DNA (1 : 10), (5) Tf-PEI/PEI25/DNA (1 : 20), (6) ligandfree PEI22/DNA, (7) Tf-PEI/PEI22/DNA (1 : 5), (8) 3 mg naked DNA. Gene Therapy


gene expression was detectable in postnatally injected livers (neonate and adult) 48 h after application of either ligand-free or any of the transferrin-conjugated PEI25/ DNA complexes. The highest transgene expression values with ligandfree PEI-complexed DNA were around 3 104 RLU per fetally injected liver, corresponding to 9.8 1037 0.7 103 RLU/mg of tissue protein or approximately 20 ng luciferase per gram liver protein. These high values were seen with ligand-free PEI22/DNA generated in salt-containing buffer at N/P 6.0 (Figure 2a, lane 6). In neonatally injected livers under the same conditions, reporter gene expression decreased to 5.4 1037 0.6 103 RLU/mg tissue protein (Figure 2c, lane 6) and in adult organs to only 0.1 10370.06 103 RLU/mg liver protein (Figure 2e, lane 6). Transferrin-conjugated PEI22/DNA (ratio 1 : 5) prepared at the same ionic strength and N/P ratio revealed similar high transgene expression (9.5 10372.6 103 RLU/mg liver protein) in in utero injected animals (Figure 2a, lane 7). In the livers of postnatally injected animals, these complexes yielded up to two orders of magnitude lower expression values with 0.4 10370.1 103 RLU (Figure 2c, lane 7) and 0.06 10370.04 103 RLU/mg liver protein (Figure 2e, lane 7) in 3-day- and 8-week-old mice, respectively. Under salt-free conditions, transferrin-coupled PEI22/ DNA (ratio 1 : 5) at N/P 6.0 induced luciferase expression up to 7.3 10371.9 103 RLU/mg liver protein in prenatally injected mice, whereas in 3-day-old neonates transgene expression with 1.2 10370.3 103 RLU/mg liver protein was six-fold lower than after intrauterine injection. No luciferase expression was measurable in adult organs (data not shown). Administration of the same amount of naked DNA into fetal livers as used in the various PEI/DNA complexes (3 mg) yielded 11-fold lower reporter gene expression values 48 h postinjection (0.9 10370.4 103 RLU/mg liver protein (Figure 2a, lane 8)). In neonatally injected livers, the values were also very low with 0.6 10370.2 103 RLU/mg liver protein (Figure 2c, lane 8). In the adult livers injected, however, no expression could be detected with this amount of DNA. Increasing the concentration of naked DNA to 9 mg increased reporter gene expression in fetally injected mouse livers to 3.2 10471 104 RLU/mg liver protein (Figure 3a). In neonatal and adult livers, reporter gene expression values were approximately 2- and 40-fold lower than in the prenatally injected mice, respectively (1.8 10470.4 104 RLU and 0.8 10370.2 103 RLU/mg tissue protein (Figure 3a)).

Transgene expression in nontarget organs To determine whether the complexes injected into fetal, neonatal or adult mouse livers remain within the targeted organ or are distributed beyond the site of application to other organs, luciferase expression was analyzed in lungs, kidneys and gonads of the animals injected. In most cases PEI25-based complexes yielded no detectable luciferase expression in the fetal lungs as the organs next in circulation. However, with transferrinconjugated PEI25/DNA (ratio 1 : 4) in 0.5 HBS at N/P 6.0 low values of 0.2 10370.2 103 RLU/mg lung protein were measured (Figure 2b, lane 2). In animals

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Figure 3 Luciferase expression in mouse livers and lungs after administration of naked DNA. Luciferase expression (RLU) per milligram tissue protein in livers (a) and lungs (b) of mice of different ages after direct intrahepatic injection of increasing amounts of naked DNA. Transgene expression was measured by luciferase assay 48 h after DNA administration. Samples where no luciferase expression was detected are indicated as n.d.

injected postnatally with these complexes (3-day- or 8-week-old), no expression was detected in the lungs. Transferrin-conjugated PEI22/DNA complexes (ratio 1 : 5) generated in salt-free conditions (N/P 6.0) showed reporter gene expressions of 0.7 10370.2 103 RLU/mg lung protein in prenatally injected mice, 0.23 1037 0.04 103 RLU/mg lung protein in neonatally injected animals and only 0.17 10370.06 103 RLU/mg tissue protein in adult lungs, while complexes formed in 0.5 HBS induced transgene expression values in mouse lungs, which were generally lower than seen under salt-free conditions (Figure 2b, d and e, lanes 7). With ligand-free PEI22/DNA in 0.5 HBS at N/P 6.0, only 0.4 10370.2 103 RLU/mg lung protein was detected after intrauterine gene delivery (Figure 2b, lane 6). In neonatally injected mice 0.3 10370.1 103 RLU/mg lung protein (Figure 2d, lane 6), and in adult animals an average of 0.1 103 RLU/mg tissue protein (Figure 2f, lane 6) was observed. Administration of 3 mg naked DNA into fetal livers resulted in two-fold higher luciferase expression in the livers than in the lungs (Figure 2a and b, lanes 8). In the lungs of animals injected as neonates, reporter gene expression values were three-fold lower than the values observed in the livers (Figure 2c and d, lanes 8), and in adults no transgene expression was seen with this low Gene Therapy


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DNA concentration. However, the application of concentrated naked DNA (9 mg) into fetal livers induced an increase of reporter gene expression in the lungs (2.2 10370.2 103 RLU/mg lung protein) that paralleled the increase of luciferase expression in the target organ (3.2 10471 104 RLU/mg liver protein). This was similar for neonates, where 1.5 10370.2 103 RLU/mg lung protein was detected. Injections of the same amount of naked DNA (9 mg) into adult livers showed only trace amounts of luciferase expression in the corresponding lungs (0.04 10370.02 103 RLU/mg tissue protein (Figure 3a and b)). In general, reporter gene expression in the lungs, as the organ next to the liver in blood circulation, was 10–20 fold lower as compared to gene expression in the liver. The only exception was found with PEI22/DNA complexes in adult animals, where similar or even higher gene expression was found in the lungs compared to the liver, which is in agreement with previous studies showing particularly high gene transfer into lungs with PEI22/DNA complexes.15,18,19 No reporter gene expression was detected in the kidneys or gonads of any of the mice analyzed (266 mice injected prenatally with different PEI/DNA complexes; 49 mice injected prenatally with different amounts of naked DNA and 78 postnatally injected mice (3-day-old and 8-week-old) with either PEI-complexed or naked DNA).

Detection of in utero transduced liver cells After successful gene transfer into fetal mouse livers as determined by luciferase gene expression, we analyzed the cells responsible for transgene expression by administering a b-galactosidase reporter gene as naked DNA and within ligand-free PEI22/DNA complexes (generated in 0.5 HBS at N/P 6.0) into livers of fetal mice at E17.5. Gene expression was seen in single cells and small cell clusters randomly distributed throughout the liver (Figure 4a and b). The reporter-gene-expressing cells were polygonally shaped with a wide cytoplasm as typical for hepatocytes, but additional targeting of hematopoietic or sinusoidal cells cannot be excluded. Furthermore, standard histological staining of the sections showed no signs of major inflammation or necrosis in the injected livers. Control animals injected with 0.5 HBS or HBG were constantly negative for bgalactosidase expression.

Discussion Intrauterine gene transfer with a series of different viral vectors has been investigated previously with variable success.4–7 In contrast to gene transfer into adult individuals, for successful gene delivery into fetuses, additional parameters become critical and need to be taken into consideration. They include, among others, the very limited volume applicable, and the effects of both gene transfer procedure and utilized vectors on pregnancy. In the present work, the influence of the physical and chemical properties of PEI/DNA complexes on gene transfer efficacy into livers of fetal mice and on the survival of the treated individuals was studied. Among the PEIs tested, PEI800 appeared not to be applicable for in utero gene delivery. PEI800/DNA particles, although quite effective in vitro, yielded no transgene expression in vivo (for comparison of in vitro and in vivo gene transfer efficacies, see Figures 1 and 2). With increasing N/P ratios even marked fetal mortality was observed, most probably caused by embolic events either in the livers or lungs of the injected animals because of the high electropositivity of the complexes leading to aggregation with blood components.15 According to our results, linear PEI22 is better suited for in utero application than the branched forms of the polycation (PEI800 or PEI25). Best in vivo results were obtained with ligand-free PEI22/DNA complexes generated in the presence of salt. These complexes induced highest local transgene expression values in prenatally injected livers (mean 9.8 103 RLU/mg liver protein) and were very well tolerated by the fetuses treated (survival rate of 90%). Coupling of transferrin to PEI22/ DNA also led to high reporter gene expression in the targeted livers, but the low fetal survival (38% in 0.5 HBS and 25% in HBG) did not suggest any advantage of this coupling strategy for in utero applications. This was surprising, since incorporation of the ligand into the complexes had previously been shown to be beneficial for postnatal in vivo approaches.15 Previous reports have described much higher reporter gene expression in adult mouse lungs with salt-free PEI22/DNA complexes as compared to salt-containing formulations.18,19 This was confirmed in our study. Luciferase expression in the lungs of the PEI22-injected

Figure 4 Visualization of the in vivo transfected cells. b-galactosidase expression in liver cells of in utero injected mice (at E17.5). (a) Overview picture of a mouse liver section 48 h after application of ligand-free PEI22-complexed DNA (generated in 0.5 HBS at N/P 6.0) directly into the fetal liver. The blue staining corresponds to the cytoplasmic transgene expression as verified by lacZ staining. Reporter gene expressing liver cells are arranged individually or in small clusters. (b) High magnification of a cluster of liver cells transfected with ligand-free PEI22/DNA complexes under salt conditions and at N/P 6.0. Gene Therapy


mice (fetal, neonatal or adult) was highest with transferrin-conjugated particles generated in salt-free buffer. In salt-containing solutions, however, the same complexes induced very low expression values. This is probably because of the different sizes of the particles. Salt-free PEI22 particles are small and can easily traverse endothelial cells of lung capillaries to reach alveolar cells and pneumocytes.19 PEI22/DNA particles formed in salt solutions, however, possess an inherent kinetic instability under salt conditions.19 Over a brief period of time they grow to large aggregates that associate with cells,19 thereby remaining in the target organ, where they induce local reporter gene expression. This underlines the importance of the size and aggregation status of PEIcomplexed DNA in addition to factors like surface charge of the particles or the structure of the PEI used (for examples of different sizes and aggregation statuses of the various complexes see, Figure 1). With PEI25, best results for in utero administration were obtained after incorporation of transferrin at high densities into the complexes, thereby reducing the highly positive surface charge of the particles. Owing to reduced nonspecific interactions with blood components, for example, with erythrocytes or cells of the reticuloendothelial system, transferrin-coupled complexes are better tolerated in vivo.15 In our experiments this was reflected in increased survival with increasing transferrin concentration. In accordance with previous work19 luciferase expression values with PEI25-based complexes were generally lower in the livers or lungs of all treated mice (fetal, neonatal or adult) than with PEI22/DNA. Despite the overall low reporter gene expression levels obtained with PEI-based formulations, which were far below therapeutic relevance even with the most efficient complexes (ligand-free PEI22/DNA), these nonviral vectors could be interesting for in utero gene delivery for research purposes, particularly because PEI is devoid of side-effects associated with viral vectors. However, for therapeutic applications substantial further optimization will be necessary. Administration of higher concentrated PEI/DNA complexes into fetal mouse livers was not possible, since increasing the amount of DNA to more than 3 mg at the same time would require an increase of both the polycation and the volume of the solvent. However, by using naked DNA higher amounts of DNA can be administered while keeping the injection volume constant. The applicability of naked DNA for postnatal gene delivery into various tissues has been shown previously in several studies,20–22 but to our knowledge no detailed work on intrauterine application of naked DNA has been reported to date. Administration of the same amount of naked DNA as used in the PEI/DNA complexes (3 mg) revealed 11-fold lower luciferase expression values than the highest values measured with complexed DNA. However, higher concentrations of naked DNA (6 and 9 mg per 7 ml) provided higher luciferase expression levels. The values we obtained with 9 mg naked DNA, the highest concentration of DNA administered in this study, were almost 40 times higher in the livers of fetal mice as compared to adult livers, suggesting that, in comparison to adults, fetal tissue seems to be particularly amenable to the uptake and expression of naked DNA. This could be a consequence of several factors: (i) protection of the injected DNA from DNase

degradation, (ii) facilitated DNA passage through liver sinusoidal fenestrae via rapid injection at a pressure higher than fetal blood pressure,23,24 (iii) more efficient uptake of naked DNA into fetal hepatocytes because of the higher cell division rate in fetuses than in adults,25 and (iv) reduced activity of cytosolic nucleases in fetal cells26–28 allowing the extended intracellular persistence of naked DNA, a requirement for augmented transgene expression.29,30 In conclusion, the primary goal of this study was to evaluate in a short-term study critical parameters for gene delivery into fetal mouse livers with PEI-complexed DNA. We found that even with the most suitable complexes (ligand-free PEI22/DNA) the levels of gene expression achieved in the target organ were clearly below therapeutic relevance. Surprisingly, fetal liver appeared to be particularly accessible to uptake and expression of naked DNA. This observation can be regarded as a basis for further exploration of naked DNA applicability for genetic modification of fetal tissues, especially since, to date the use of naked DNA has not been observed to be associated with serious sideeffects.31–33

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Materials and methods Chemicals Branched PEIs, molecular weights 25 and 800 kDa, were obtained from Aldrich (Milwaukee WI, USA); and Fluka (Buchs, Switzerland) (50% w/v), respectively. Linear PEI (22 kDa) was purchased from Euromedex (Soufferlweyersheim, France) or, more recently, from MBI Fermentas (St Leon-Rot, Germany). Transferrin–PEI conjugate synthesis was performed as described previously.15. Plasmids The plasmids pCMVL coding for the Photinus pyralis luciferase gene and pCMVb coding for b-galactosidase were purified using the Endofree Plasmid Giga Kit (Qiagen, Hilden, Germany). Endotoxin (LPS) levels were o0.1 endotoxin units/50 mg DNA as determined by Limulus Amebocyte Lysate assay (BioWhittaker, Walkersville, MD, USA). PEI/DNA complex formation Complexes were prepared essentially as described previously.34 Indicated amounts of plasmid DNA and PEI were each diluted at different N/P ratios (4.8, 6.0 or 7.2) to a final volume of 250 ml with either 0.5 HBS (75 mM NaCl, 20 mM HEPES pH 7.4 + 5% glucose) or HBG (20 mM HEPES pH 7.4+5% glucose), mixed thoroughly by pipetting and incubated for 20 min at room temperature to allow complex formation. Electron microscopy of polycation/DNA complexes For negative staining, 0.4% formvar-coated grids were used. Complex solutions were adsorbed on the grids for 10 min. Particles were fixed in 2.5% glutaraldehyde/ 0.1 M cacodylate buffer pH 7.3 for 1 min, washed four times with distilled water, stained with 1% uranyl acetate for 1 min and examined in a Philips CM 100 electron microscope. Gene Therapy


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Transfection of cultured cells At 24 h before transfection, 3 105 3T3 cells were seeded in 60 mm plates (Costar, Cambridge, MA, USA). Transfections were performed with 9 mg complexed DNA mixed with 1.5 ml culture medium (DMEM containing 10% fetal bovine serum). After 4 h incubation, transfection medium was replaced with 2 ml of fresh culture medium. Mouse experiments Mice. B6/CBA/F1-hybrid mice were purchased from Harlan Winkelmann (Borchen, Germany). The day of vaginal plug appearance was counted as day 0.5 of gestation (E 0.5). Injection of the complex solutions into livers of mice at different ages. (i)

Pregnant mice were anesthetized by an intraperitoneal injection of Avertin (2.5% tribromoethanol in tert-amyl alcohol diluted in phosphate-buffered saline (PBS)) (Sigma-Aldrich, Seelze, Germany). The abdomen was opened by ventral midline laparatomy and one uterine horn was exposed. An aluminosilicate capillary was filled with the injection solution for direct transuterine administration into the fetal livers. The same procedure was performed in the fetuses of the second horn. Finally, abdominal wall and skin were closed separately by surgical sutures (Ethicon 3-0, Norderstedt, Germany). (ii) Neonatal mice, 3-day-old, were briefly anesthetized by inhalation of Forane (Isoflurane, Abbot, Queenborough, UK). Injection solution was administered directly through the skin into the liver. (iii) Adult mice, 8-week-old, were anesthetized intraperitoneally with Avertin. Their livers were exposed after the incision of skin and peritoneum along the right costal arch. Injection solution was administered directly into the right central lobe of the liver. The abdominal wall was closed in two layers with surgical sutures (Ethicon 3-0).

Harvesting of animal organs for analysis of transgene expression. The organs (liver, lungs, kidneys and gonads) of all injected mice were harvested 48 h after the gene transfer procedure. At this time point the treated fetuses had already undergone parturition. Newborn animals were killed by decapitation and adult animals by cervical dislocation. The animals were immediately dissected, their organs harvested, rinsed in 41C PBS solution and snap-frozen in isopentane at the temperature of liquid nitrogen. Detection of reporter gene expression (i) Luciferase activity. Frozen mouse organs were thawed in a 371C water bath and placed on ice during the next steps. After addition of 600 ml lysis buffer (25 mM Tris phosphate pH 7.8, 2 mM DTT, 2 mM EDTA, 10% glycerol, 1% Triton X-100) to the fetal and neonatal organs and 900 ml of the buffer to the adult organs, the organs were homogenized and immediately frozen in liquid nitrogen. The specimens were thawed again and placed on ice. To pellet, cell debris was centrifuged for 10 min at 41C and Gene Therapy

15 000 rpm. Luciferase activity in the tissues was finally recorded from 50 ml of the supernatants by luminometry (Lumat LB9507 instrument/EG u. G Berthold, Bad Wildbach, Germany). Background signal (130–250 RLU) was subtracted from each value. Luciferase levels representing transfection efficacy were measured as RLU per organ or milligram tissue protein. According to the luciferase standard used, 1 106 RLU correspond to approximately 2 ng luciferase. (ii) Protein assays. To normalize luciferase expression to the average weight of mouse livers, the protein contents of fetal (E17.5), 3-day-old neonatal and 8week-old adult livers and lungs were measured using Bradford assay.35 The protein contents of mouse livers at the different ages were as a mean: fetal 3.4 mg, neonatal 4.7 mg and adult 76 mg. The corresponding values for mouse lungs were: fetal 2.7 mg, neonatal 3.2 mg and adult 10.4 mg. (iii) b-Galactosidase assay. At 24 h after transfection, the cultured cells were washed with PBS, fixed for 10 min in 0.5% glutaraldehyde/PBS at 371C, again washed with PBS and incubated for 4 h at 371C in Xgal staining solution.36 For in vivo detection of b-galpositive cells serial cryosections of the frozen fetuses were prepared and incubated overnight at 371C in X-gal staining solution. Before mounting, the sections were counterstained with eosin.

Acknowledgements We thank Andrea Fuchsbichler for performing electron microscopy, Vanessa Ro¨ssler for technical help with luciferase measurement and Dr Charles Buck for critically reading the manuscript. This work was supported by grants from the Austrian Science Fund (S 7401- MOB to KZ) and the Austrian Federal Ministry for Social Security and Generations (to KZ).

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