Molecular Genetics and Metabolism 82 (2004) 20–26 www.elsevier.com/locate/ymgme
Non-viral delivery of the porphobilinogen deaminase cDNA into a mouse model of acute intermittent porphyria Annika Johansson,a,¤ Grzegorz Nowak,b Christer Möller,c and Pauline Harpera a
Porphyria Centre Sweden, Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden b Division of Transplantation Surgery, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Stockholm, Sweden c HemeBiotech A/S, Lidingö, Sweden Received 23 January 2004; accepted 27 February 2004
Abstract Acute intermittent porphyria (AIP), an inborn error of metabolism, results from the deWcient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyria include episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenous heme administration is palliative and there is no way to prevent the attacks. Thus, eVorts are focused on methods to replace the deWcient activity in the liver to prevent the acute attacks of this hepatic porphyria. Here we explore the eYciency of a non-viral gene delivery to obtain PBGD expression in the liver of AIP transgenic mice. Four vectors were evaluated: naked DNA and DNA complexed to liposomes, polyethylenimine (PEI), and PEI-galactose, using a luciferase construct as reporter gene. The vectors were administered intravenously or directly into the portal vein with transient blood Xow blockage. After tail vein injection of the DNA complexes, the liposome vector had the highest luciferase expression in lung and less in liver. When injected into the portal vein, the naked DNA had considerably higher hepatic reporter gene expression; 100 g of naked DNA had the highest hepatic luciferase expression 24 h after portal vein injection. When these vectors were used to deliver the PBGD gene into the AIP mouse model no enhancement of the endogenous PBGD activity in liver was detectable, despite the presence of the PBGD-plasmids as veriWed by PCR. Thus, more eYcient non-viral vectors are needed to express suYcient PBGD activity over the endogenous hepatic level (»30% of normal) in this murine system. 2004 Elsevier Inc. All rights reserved. Keywords: Acute intermittent porphyria; Galactose; Liposomes; Luciferase; Non-viral gene delivery; Polyethylenimine; Porphobilinogen deaminase
Introduction Acute intermittent porphyria (AIP), an autosomal dominant hepatic porphyria, results from insuYcient activity of the third enzyme in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD, EC 4.3.1.8). This is the most frequent and severe form of the acute porphyrias [1]. Clinical symptoms include acute intermittent attacks of abdominal pain, peripheral neuropathy, and psychiatric disturbances [2]. The current treatment of AIP, based on heme replacement and car¤
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[email protected] (A. Johansson).
1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.02.008
bohydrate loading [3], is only palliative and there is no treatment to prevent the life-threatening acute attacks. Gene therapy has been proposed as a potential cure and non-viral vectors have been used for in vitro delivery of the PBGD cDNA to obtain high levels of PBGD activity [4], which can function in the heme biosynthetic pathway [5]. The next step would be to Wnd a gene delivery system that expresses PBGD in vivo with the liver as the main target organ, as this is a major site of heme synthesis [2]. Thus, gene delivery of suYcient hepatic PBGD activity may totally improve the disease, as has been the result of the recent liver transplantation in a female AIP heterozygote whose chronic neurologic attacks ceased after transplantation [6].
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Several gene delivery systems have been used to transfer genes to the liver using both viral and nonviral vectors [7]. Although the non-viral vectors are less eYcient than the viral, the synthetic delivery systems continue to attract interest because of their safety and low immunogenicity proWles [8]. Expression of transgenes in mouse liver has been achieved by using the hydrodynamic procedure, which is based on a rapid injection of a large volume of naked DNA into the tail vein [9–11] or into the portal vein [12]. Alternatively, eYcient gene transfer of naked DNA to the liver without the hydrodynamic pressure has been demonstrated by transiently restricting hepatic blood Xow following intravenous injection of a small volume containing the vector [13]. In addition, cationic liposomes have been shown to deliver transgenes to the liver after portal vein injection whereas tail vein injection almost always resulted primarily in expression in lung [14,15]. The cationic polymer, polyethylenimine (PEI), has also delivered transgenes in vivo to several organs such as kidney [16], lung [17], and liver [18,19]. A recent improvement of the PEI technology is galactosylated PEI (PEI-Gal) that has been used for targeting the liver [20]. Galactose attaches to the asialoglycoprotein receptor that is speciWcally expressed on hepatocytes [21]. In this communication, we describe eVorts to evaluate non-viral hepatic gene delivery in an AIP mouse model that is partially deWcient in PBGD [22]. Four vectors were evaluated using luciferase as reporter enzyme: naked DNA, DNA complexed to liposomes, PEI, and PEI-Gal. The eYciency of these vectors for hepatic gene delivery were evaluated following tail vein and portal vein injection with transient blood-Xow blockage. These studies provide a system to evaluate eVorts to increase hepatic PBGD activity in the murine AIP model using gene delivery strategies.
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Preparation of DNA complexes The plasmids were used naked or complexed to liposomes, polyethylenimine (PEI) or PEI-galactose (PEIGal). Naked plasmid DNA was dissolved in sterile saline (0.9% NaCl). The liposome formulation in vivo GeneSHUTTLE (Qbiogene, Carlsbad, CA, USA) was condensed to the plasmid DNA according to the manufacturer’s protocol. For the tail vein injections, the liposomes were prepared to a Wnal concentration of 4 mmol/L, and for the portal vein injections to 1.6 mmol/ L. The liposomes consist of the cationic lipid DOTAP (1,2-bis[oleoyloxy]-3-[trimethylammonio]propane) and cholesterol at a 1:1 molar ratio. To form the PEI/DNA complexes, the plasmid DNA was Wrst added to a 5% glucose solution. A working stock solution of branched PEI 25 kDa (10 mmol/L, pH 7.0) (Aldrich, St. Louis, MO, USA) was passed through 0.22 m Wlters (Millipore, Bedford, MA, USA) and added to the plasmid DNA solution to yield an N/P ratio of 10 [23]. The ratio is based on the fact that 1 g DNA corresponds to 3 nmol of phosphate (P) and 1 L of PEI stock solution contains 100 nmol of amino nitrogen (N) [23]. The galactose-conjugated linear PEI (22 kDa) (In vivo-jetPEIGal) was purchased from PolyPlus-transfection (Illkirch, Cedex, France). Plasmid DNA was diluted in 5% glucose and the in vivo-jetPEI-Gal solution was added to form PEI-Gal/DNA complexes with an N/P ratio of 10, according to the manufacturer. Animals Adult female and male wild type (C57BL/6) and AIP mice were used in this study. The generation of the AIP mouse model was performed as described by Lindberg et al. [22]. The local Ethics Committe approved all animal procedures used in this study. The mice were treated in accordance with the Swedish regulations and laws for care and use of laboratory animals.
Materials and methods Injection procedures Expression plasmids The pEGFPLuc plasmid, which expresses the WreXy luciferase, was obtained from Clontech (Palo Alto, CA, USA). The p-mPBGDhou plasmid, which expresses mouse housekeeping PBGD activity from the cytomegalovirus (CMV) immediate early promoter/enhancer, was constructed as previously described [4]. Both plasmids were expanded in Escherichia coli DH5 (Gibco, Grand Island, NY, USA) and puriWed using the Qiagen Plasmid Maxi Kit (Qiagen, Chatsworth, CA, USA). Purity was conWrmed by the absorbance measurement at 260 and 280 nm, and the absence of RNA and genomic DNA was evaluated by agarose gel electrophoresis. Aliquots of plasmid DNA were stored at ¡20 °C.
The DNA complexes were injected in standard way into the tail vein in a volume of 200 L. For portal vein injections, the mice were anesthetized with isoXurane (Fluovac Unit, IMS, Chechire, UK) and the liver was exposed through a ventral midline incision. Prior to intraportal injection, microvessel clamps (S & T, Neuhausen, Switzerland) were placed at the portal vein, the supra- and infrahepatic vena cava and injection was performed above the clamp on the portal vein. The DNA complexes were injected in a volume of 500 L over approximately 40 s using a 30-gauge needle and 1 mL syringe. After the intraportal injections the needle was removed and back Xow was prevented by pressure with a cotton wool bud. Two minutes after the injection
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procedure was Wnished the microvessel clamps were removed and the blood Xow through the liver was restored.
the blank sample from each tissue was subtracted. The PBGD activity was expressed as pkat per gram tissue protein.
Tissue preparation
Detection of plasmid DNA by PCR
At time-points after the injections (8, 24, 48, 96, and 144 h), the animals were anesthetized with isoXurane before blood was collected in MiniCollect tubes with Liheparin additive (Greiner Bio-One, Longwood, FL, USA). The liver, kidney, spleen, lung, and brain were harvested. The tissues were immediately frozen in liquid nitrogen and stored at ¡80 °C until preparation. To measure the luciferase activity, the tissues were placed in 1 mL of 1£ Cell Culture Lysis Reagent (Promega, Madison, WI, USA). The liver was divided in four parts before homogenization. Each sample was homogenized on ice, using a Potter–Elvehjelm glass homogenizer (inner diameter 8.0 mm, frosted walls) Wtted with a TeXon pestle (diameter 7.8 mm), at a speed of 120 rpm. The sample was centrifuged at 10,000g for 10 min (4 °C) and the supernatant was stored at ¡80 °C until analysis. The liver homogenate was pooled in one test-tube. To measure the PBGD activity, the tissues were placed in 2 mL of 50 mmol/L Tris–HCl buVer, pH 8.2, and homogenized using the procedure described above.
Polymerase chain reaction (PCR) analysis was performed to evaluate the time course of plasmid DNA clearance from the liver. Total DNA was isolated from liver tissue using a QIAamp DNA MiniKit (Qiagen) according to the tissue protocol. Approximately 5 ng of DNA was subjected to the PCR analysis using primers speciWc for the CMV promoter region. The upstream and downstream primers were 50-GGT CAT TAG TTC ATA GCC C-30 and 50-GAT GTA CTG CCA AGT AGG-30, respectively. The expected size of the ampliWed product was 310 bp. The PCR reaction was carried out in a total volume of 50 L of 1.5 mmol/L MgCl2, 125 mol/L dNTP’s, 20 pmol of each primer, and 1 unit of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA). AmpliWcation was arrested after 25 cycles (94 °C £ 30 s, 56 °C £ 30 s, and 72 °C £ 30 s). The samples were subjected to agarose gel electrophoresis (1%) and visualized with ethidium bromide.
Luciferase assay
Results
The luciferase activity was measured using the Luciferase Assay System (Promega). Tissue homogenate (20 L) was added to 100 L of luciferase substrate and the relative light units (RLU) were measured over 10 s in a multi-well luminometer (1420 Victor2 Multilabel Counter, Wallac, Finland). The samples were analyzed in duplicates and the background signal (110–350 RLU) was subtracted. PuriWed recombinant luciferase (Promega) was used to produce a standard curve of RLU versus amount of enzyme. According to the luciferase standard used, 1 £ 106 RLU corresponds to approximately 1 ng luciferase. Protein content was determined using a Micro BCA Protein Assay Kit (Pierce, Rockford, Illinois, USA) and the luciferase activity was expressed in terms of RLU per milligram tissue protein.
Expression of luciferase after tail vein injection of DNA complexes
PBGD activity assay
Expression of luciferase after portal vein injection of DNA complexes
The PBGD activity was assayed by measuring the conversion of PBG to uroporphyrin according to the method of Magnussen et al. [24]. The protein content was analyzed using the Bio-Rad DC Protein Assay (Hercules, CA, USA), and the samples were diluted to a protein content of 0.5 g/L with 50 mmol/L Tris–HCl buVer, pH 8.2. A volume of 1.45 mL of diluted sample was used in the assay, performed as previously described [4]. The assay was performed in duplicates and the intensity of
Following tail vein injection of 100 g of the pEGFPLuc vector as naked DNA or complexed to liposomes, polyethylenimine (PEI) or galactose conjugated PEI (PEI-Gal), the activity of luciferase was determined in liver, lung, kidney, spleen, and brain in wild type (C57BL/6) mice (Fig. 1). The highest level of luciferase expression after 24 h was found in lung using the liposomes with lower levels in the other tissues. Using PEI or PEI-Gal, the highest luciferase activity was also found in lung with barely detectable levels in the other tissues. Injection of naked DNA resulted in very low or undetectable levels of luciferase expression in all tissues.
To improve transgene expression in liver, 100 g of pEGFPLuc, naked or complexed to liposomes, PEI or PEI-Gal, were injected into the portal vein with transient blood Xow blockage. At 24 h after injection, the highest luciferase expression was found in liver using naked DNA with lower expression in the other tissues (Fig. 2). Using PEI and PEI-Gal, higher luciferase expression levels in liver were also observed as com-
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Fig. 1. Luciferase activity in wild type (C57BL/6) mice injected in tail vein with 100 g of the luciferase expression plasmid (pEGFPLuc), naked or complexed to cationic liposomes, polyethylenimine (PEI) or galactose conjugated PEI (PEI-Gal), in a volume of 200 L. The luciferase activity, expressed as RLU/mg protein, was analyzed in liver, lung, kidney, spleen, and brain tissue homogenate 24 h after injection. Data are presented as means of two animals.
Fig. 2. Luciferase activity in wild type (C57BL/6) mice injected in portal vein, with transient blood Xow blockage, with 100 g of the luciferase expression plasmid (pEGFPLuc), naked or complexed to cationic liposomes, polyethylenimine (PEI) or galactose conjugated PEI (PEI-Gal), in a volume of 500 L. The luciferase activity, expressed as RLU/mg protein, was analyzed in liver, lung, kidney, spleen, and brain tissue homogenate 24 h after injection. Data are presented as means of two animals.
pared with tail vein injection. Using liposomes, low luciferase expression was found in all tissues and areas of extensive hepatic necrosis were found at autopsy in these animals. DNA dose dependence on luciferase expression The eVect of the amount of naked pEGFPLuc (25– 400 g) on luciferase expression was studied in wild type mice 24 h after portal vein injection. The highest level of luciferase expression in liver was obtained after injection of 100 g of pEGFPLuc (Fig. 3).
Expression of PBGD after portal vein injection of naked DNA The p-mPBGDhou vector was injected as naked DNA (100 g) into the portal vein of the AIP mouse model, as this gave the highest transgene expression in liver when using pEGFLuc. At diVerent time-points (8– 144 h) after injection, PBGD activity was measured in liver homogenates and compared with the endogenous level in AIP mice. No increased PBGD activity was observed (Fig. 4). Only one treated AIP mouse showed increased PBGD activity up to wild type after 8 h, but
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Fig. 3. Dose–response analysis of luciferase activity versus amount of luciferase expression plasmid (pEGFPLuc) in wild type (C57BL/6) mice. The mice were injected in portal vein in combination with transient blood Xow blockage with increasing amounts of naked pEGFPLuc (25–400 g) in a volume of 500 L. The luciferase activity, expressed as RLU/mg protein, was analyzed in liver 24 h after injection. Data are presented as means of two animals.
Fig. 4. PBGD activity in liver of the AIP mice after portal vein injection with transient blood Xow blockage of 100 g of naked p-mPBGDhou, coding for the mouse housekeeping PBGD, in a volume of 500 L. The PBGD activity, expressed as pkat/g protein, was analyzed at diVerent time-points (24–144 h) after injection. As reference, the endogenous PBGD activity was measured in liver tissue from wild type and AIP mice. Data are presented as means of two animals and means § SD of 6–12 animals.
this could not be repeated (data not shown). As control, injection of physiological saline (500 L) resulted in no change in the endogenous PBGD activity (Fig. 4).
injection was compared with that of pEGFPLuc (Fig. 5) from which the highest hepatic expression was found (Fig. 2).
Detection of plasmid DNA in liver Discussion DNA was extracted from liver tissue after portal vein injections of 100 g of naked p-mPBGDhou or pEGFPLuc. The DNA was PCR-ampliWed using primers speciWc for the CMV promoter. The plasmid p-mPBGDhou was detectable in liver as early as 8 h after portal vein injection and was detectable for at least 144 h (Fig. 5). The hepatic presence of p-mPBGDhou at 24 h after
Hepatic gene therapy oVers the possibility of preventing or minimizing the severity of the acute neurologic attacks in patients with AIP. Recently, it was shown that functional PBGD can be expressed in vitro [4] and correct the biochemical defect in PBGD-deWcient cells by using non-viral vectors [5]. Here we have extended the
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Fig. 5. Detection of p-mPBGDhou and pEGFPLuc with PCR analysis. PCR ampliWcation of DNA extracted from liver tissue after portal vein injection with transient blood Xow blockage of 100 g of plasmid DNA shows a 310-bp fragment of the CMV promoter. Lane 1, size marker; lanes 2–6, various time points after portal injection of p-mPBGDhou; lane 7, the negative control from PBGD-deWcient mouse injected with 0.9% NaCl solution without plasmid construct; lane 8, negative control from a wild type mouse; lane 9, positive control p-mPBGDhou used as template; lane 10, portal injection of 100 g of pEGFPLuc after 24 h to a wild type mouse; lane 11, positive control pEGFPLuc used as template; and lane 12, blank.
non-viral gene delivery approach for hepatic PBGD expression in the AIP mouse model [22]. Four vectors were evaluated that had previously been shown to transfer genes into liver with luciferase as the reporter enzyme: naked DNA and DNA complexed to cationic liposomes, the cationic polymer polyethylenimine (PEI), and galactose-conjugated PEI (PEI-Gal) [12,14,18,20]. Luciferase expression was detected in all tissues analyzed after tail vein injections of the DNA complexes in a volume of 200 L (Fig. 1). The highest level of expression was observed in lung using liposomes, with a lower level in the liver. A similar tissue distribution of reporter gene expression using liposomes has been reported [14]. Liposomes injected in the tail vein seem to be predominantly taken up by the lungs, presumably because the pulmonary capillary system is perfused Wrst [25]. Injection of naked DNA and DNA complexed to PEI and PEI-Gal resulted in low or barely detectable levels of luciferase in all tissues analyzed (Fig. 1). The low levels of expression may result from the rapid degradation of the plasmids by serum nucleases and/or by the rapid clearance from the circulation by KupVer cells [26]. PEI, and most likely also PEI-Gal, activate the complement system [27], which could further reduce their chances of delivering the DNA to the liver. Injection of the DNA complexes by the hydrodynamic procedure, would probably give a higher expression in liver [28], but this was not permitted by the local Ethics Committe.
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To improve hepatic luciferase expression, the DNA complexes were injected (500 L) directly to the liver via the portal vein in combination with transient blood Xow blockage. Using this technique with osmotic and hydrostatic pressure, the highest hepatic luciferase expression was found using the naked DNA vector (Fig. 2). Similar results have been reported for other naked DNA vectors [12]. Portal vein injections presumably enhance DNA transfer into hepatocytes by transiently opening the fenestrae in the endothelial barrier [29]. Injection of PEIGal also resulted in higher hepatic luciferase expression as compared with that obtained with tail vein injections, but expression was 100-fold lower than that of naked DNA (Fig. 2). A minor increase in hepatic expression was found for PEI. Portal vein injection of liposomes caused extensive hepatic necrosis, which probably accounted in part for the low luciferase expression. Hepatic necrosis caused by injected liposomes has been reported [14]. A dose–response assay was undertaken to determine the amount of naked DNA that gave the highest hepatic luciferase expression after portal vein injection. Previous dose–response studies reported that plasmid uptake or hepatic expression appeared to be limited by a saturation mechanism [9,13]. This concept was supported by our results, which indicated that saturation occurred and that the highest hepatic expression level was obtained using 100 g of naked DNA (Fig. 3). Injections of higher amounts of DNA (up to 400 g) did not increase gene expression. The highest hepatic luciferase expression resulted from injection of 100 g of naked DNA into the portal vein. When these conditions were used to inject the PBGD plasmid into the AIP mouse model, no increased PBGD activity was detected (Fig. 4), apart from one exception. At 8 h after injection, one AIP mouse had increased activity, but further trials failed to conWrm this observation. Since PBGD activity is about 30% of normal in the AIP mouse, the PBGD expressed by the vector would have to be expressed at a signiWcantly higher level to clearly elucidate its expression. Since luciferase is not endogenously expressed, it was therefore easily detected. The presence of the PBGD-plasmid in liver at the diVerent time-points was veriWed by PCR analysis of the CMV promoter (Fig. 5) and compared to the presence of pEGFPLuc in liver 24 h after portal vein injection. In conclusion, the most eYcient non-viral gene delivery system for hepatic luciferase expression was portal vein injection of naked DNA. However, gene delivery in the AIP mouse model using naked PBGD-plasmids resulted in no detectable enhancement of endogenous hepatic PBGD activity. Thus, more eYcient non-viral gene delivery systems must be developed to improve hepatic gene delivery for the treatment of AIP and other metabolic diseases in which hepatic gene therapy would provide clinical beneWt.
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