Non-viral delivery of the porphobilinogen deaminase cDNA into a mouse model of acute intermittent porphyria

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Non-viral delivery of the porphobilinogen deaminase cDNA into a mouse model of acute intermittent porphyria
  Molecular Genetics and Metabolism 82 (2004) 20–$ - see front matter ©  2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ymgme.2004.02.008 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 Harper a 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 de W cient activity of the third enzyme in theheme biosynthetic pathway, porphobilinogen deaminase (PBGD). Clinical symptoms of this autosomal dominant hepatic porphyriainclude episodic acute attacks of abdominal pain, neuropathy, and psychiatric disturbances. Current therapy based on intravenousheme administration is palliative and there is no way to prevent the attacks. Thus, e V  orts are focused on methods to replace the de W -cient activity in the liver to prevent the acute attacks of this hepatic porphyria. Here we explore the e Y ciency of a non-viral genedelivery to obtain PBGD expression in the liver of AIP transgenic mice. Four vectors were evaluated: naked DNA and DNA com-plexed to liposomes, polyethylenimine (PEI), and PEI-galactose, using a luciferase construct as reporter gene. The vectors wereadministered intravenously or directly into the portal vein with transient blood X ow blockage. After tail vein injection of the DNAcomplexes, the liposome vector had the highest luciferase expression in lung and less in liver. When injected into the portal vein, thenaked DNA had considerably higher hepatic reporter gene expression; 100  g of naked DNA had the highest hepatic luciferaseexpression 24h after portal vein injection. When these vectors were used to deliver the PBGD gene into the AIP mouse model noenhancement of the endogenous PBGD activity in liver was detectable, despite the presence of the PBGD-plasmids as veri W ed byPCR. Thus, more e Y cient non-viral vectors are needed to express su Y cient 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 autosomaldominant hepatic porphyria, results from insu Y cientactivity of the third enzyme in the heme biosyntheticpathway, porphobilinogen deaminase (PBGD, EC4.3.1.8). This is the most frequent and severe form of theacute porphyrias [1]. Clinical symptoms include acuteintermittent attacks of abdominal pain, peripheral neu-ropathy, and psychiatric disturbances [2]. The currenttreatment of AIP, based on heme replacement and car-bohydrate loading [3], is only palliative and there is notreatment to prevent the life-threatening acute attacks.Gene therapy has been proposed as a potential cure andnon-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 W nd a gene delivery systemthat expresses PBGD in vivo with the liver as the maintarget organ, as this is a major site of heme synthesis [2].Thus, gene delivery of su Y cient hepatic PBGD activitymay totally improve the disease, as has been the result of the recent liver transplantation in a female AIP hetero-zygote whose chronic neurologic attacks ceased aftertransplantation [6]. ¤ Corresponding author. Fax: +46-8-585-827-60. E-mail address: (A. Johansson).  A. Johansson et al. / Molecular Genetics and Metabolism 82 (2004) 20–26  21 Several gene delivery systems have been used totransfer genes to the liver using both viral and non-viral vectors [7]. Although the non-viral vectors are lesse Y cient than the viral, the synthetic delivery systemscontinue to attract interest because of their safety andlow immunogenicity pro W les [8]. Expression of trans-genes in mouse liver has been achieved by using thehydrodynamic procedure, which is based on a rapidinjection of a large volume of naked DNA into the tailvein [9–11] or into the portal vein [12]. Alternatively,e Y cient gene transfer of naked DNA to the liver with-out the hydrodynamic pressure has been demonstratedby transiently restricting hepatic blood X ow followingintravenous injection of a small volume containing thevector [13]. In addition, cationic liposomes have beenshown to deliver transgenes to the liver after portalvein injection whereas tail vein injection almost alwaysresulted primarily in expression in lung [14,15]. Thecationic polymer, polyethylenimine (PEI), has alsodelivered transgenes in vivo to several organs such askidney [16], lung [17], and liver [18,19]. A recentimprovement of the PEI technology is galactosylatedPEI (PEI-Gal) that has been used for targeting the liver[20]. Galactose attaches to the asialoglycoproteinreceptor that is speci W cally expressed on hepatocytes[21].In this communication, we describe e V  orts to evaluatenon-viral hepatic gene delivery in an AIP mouse modelthat is partially de W cient in PBGD [22]. Four vectorswere evaluated using luciferase as reporter enzyme:naked DNA, DNA complexed to liposomes, PEI, andPEI-Gal. The e Y ciency of these vectors for hepatic genedelivery were evaluated following tail vein and portalvein injection with transient blood- X ow blockage. Thesestudies provide a system to evaluate e V  orts to increasehepatic PBGD activity in the murine AIP model usinggene delivery strategies. Materials and methods Expression plasmids The pEGFPLuc plasmid, which expresses the W re X yluciferase, was obtained from Clontech (Palo Alto, CA,USA). The p-mPBGDhou plasmid, which expressesmouse housekeeping PBGD activity from the cytomega-lovirus (CMV) immediate early promoter/enhancer, wasconstructed as previously described [4]. Both plasmidswere expanded in Escherichia coli   DH5   (Gibco, GrandIsland, NY, USA) and puri W ed using the Qiagen PlasmidMaxi Kit (Qiagen, Chatsworth, CA, USA). Purity wascon W rmed by the absorbance measurement at 260 and280nm, and the absence of RNA and genomic DNA wasevaluated by agarose gel electrophoresis. Aliquots of plasmid DNA were stored at ¡ 20°C. Preparation of DNA complexes The plasmids were used naked or complexed to lipo-somes, polyethylenimine (PEI) or PEI-galactose (PEI-Gal). Naked plasmid DNA was dissolved in sterile saline(0.9% NaCl). The liposome formulation in vivo Gene-SHUTTLE (Qbiogene, Carlsbad, CA, USA) was con-densed to the plasmid DNA according to themanufacturer’s protocol. For the tail vein injections, theliposomes were prepared to a W nal concentration of 4mmol/L, and for the portal vein injections to 1.6mmol/L. The liposomes consist of the cationic lipid DOTAP(1,2-bis[oleoyloxy]-3-[trimethylammonio]propane) andcholesterol at a 1:1 molar ratio. To form the PEI/DNAcomplexes, the plasmid DNA was W rst added to a 5%glucose solution. A working stock solution of branchedPEI 25kDa (10mmol/L, pH 7.0) (Aldrich, St. Louis,MO, USA) was passed through 0.22  m W lters (Milli-pore, Bedford, MA, USA) and added to the plasmidDNA solution to yield an N/P ratio of 10 [23]. The ratiois based on the fact that 1  g DNA corresponds to3nmol of phosphate (P) and 1  L of PEI stock solutioncontains 100nmol of amino nitrogen (N) [23]. The galac-tose-conjugated linear PEI (22kDa) (In vivo-jetPEI-Gal) was purchased from PolyPlus-transfection (Illkirch,Cedex, France). Plasmid DNA was diluted in 5% glucoseand the in vivo-jetPEI-Gal solution was added to formPEI-Gal/DNA complexes with an N/P ratio of 10,according to the manufacturer. Animals Adult female and male wild type (C57BL/6) and AIPmice were used in this study. The generation of the AIPmouse model was performed as described by Lindbergetal. [22]. The local Ethics Committe approved all ani-mal procedures used in this study. The mice were treatedin accordance with the Swedish regulations and laws forcare and use of laboratory animals. Injection procedures The DNA complexes were injected in standard wayinto the tail vein in a volume of 200  L. For portal veininjections, the mice were anesthetized with iso X urane(Fluovac Unit, IMS, Chechire, UK) and the liver wasexposed through a ventral midline incision. Prior tointraportal injection, microvessel clamps (S & T, Neu-hausen, Switzerland) were placed at the portal vein, thesupra- and infrahepatic vena cava and injection was per-formed above the clamp on the portal vein. The DNAcomplexes were injected in a volume of 500  L overapproximately 40s using a 30-gauge needle and 1mLsyringe. After the intraportal injections the needle wasremoved and back X ow was prevented by pressure with acotton wool bud. Two minutes after the injection  22 A. Johansson et al. / Molecular Genetics and Metabolism 82 (2004) 20–26  procedure was W nished the microvessel clamps wereremoved and the blood X ow through the liver wasrestored. Tissue preparation At time-points after the injections (8, 24, 48, 96, and144h), the animals were anesthetized with iso X uranebefore blood was collected in MiniCollect tubes with Li-heparin additive (Greiner Bio-One, Longwood, FL,USA). The liver, kidney, spleen, lung, and brain wereharvested. The tissues were immediately frozen in liquidnitrogen and stored at ¡ 80°C until preparation. Tomeasure the luciferase activity, the tissues were placed in1mL of 1 £  Cell Culture Lysis Reagent (Promega, Madi-son, WI, USA). The liver was divided in four partsbefore homogenization. Each sample was homogenizedon ice, using a Potter–Elvehjelm glass homogenizer(inner diameter 8.0mm, frosted walls) W tted with aTe X on pestle (diameter 7.8mm), at a speed of 120rpm.The sample was centrifuged at 10,000  g   for 10min (4°C)and the supernatant was stored at ¡ 80°C until analysis.The liver homogenate was pooled in one test-tube. Tomeasure the PBGD activity, the tissues were placed in2mL of 50mmol/L Tris–HCl bu V  er, pH 8.2, and homog-enized using the procedure described above. Luciferase assay The luciferase activity was measured using the Lucif-erase Assay System (Promega). Tissue homogenate(20  L) was added to 100  L of luciferase substrate andthe relative light units (RLU) were measured over 10s ina multi-well luminometer (1420 Victor 2  MultilabelCounter, Wallac, Finland). The samples were analyzedin duplicates and the background signal (110–350RLU)was subtracted. Puri W ed recombinant luciferase (Pro-mega) was used to produce a standard curve of RLUversus amount of enzyme. According to the luciferasestandard used, 1 £ 10 6  RLU corresponds to approxi-mately 1ng luciferase. Protein content was determinedusing a Micro BCA Protein Assay Kit (Pierce, Rockford,Illinois, USA) and the luciferase activity was expressedin terms of RLU per milligram tissue protein. PBGD activity assay The PBGD activity was assayed by measuring theconversion of PBG to uroporphyrin according to themethod of Magnussen et al. [24]. The protein contentwas analyzed using the Bio-Rad DC Protein Assay (Her-cules, CA, USA), and the samples were diluted to a pro-tein content of 0.5g/L with 50mmol/L Tris–HCl bu V  er,pH 8.2. A volume of 1.45mL of diluted sample was usedin the assay, performed as previously described [4]. Theassay was performed in duplicates and the intensity of the blank sample from each tissue was subtracted. ThePBGD activity was expressed as pkat per gram tissueprotein. Detection of plasmid DNA by PCR Polymerase chain reaction (PCR) analysis was per-formed to evaluate the time course of plasmid DNAclearance from the liver. Total DNA was isolated fromliver tissue using a QIAamp DNA MiniKit (Qiagen)according to the tissue protocol. Approximately 5ng of DNA was subjected to the PCR analysis using primersspeci W c for the CMV promoter region. The upstreamand downstream primers were 5 0 -GGT CAT TAG TTCATA GCC C-3 0  and 5 0 -GAT GTA CTG CCA AGTAGG-3 0 , respectively. The expected size of the ampli W edproduct was 310bp. The PCR reaction was carried outin a total volume of 50  L of 1.5mmol/L MgCl 2 ,125  mol/L dNTP’s, 20pmol of each primer, and 1unitof Ampli Taq  DNA polymerase (Applied Biosystems,Foster City, CA, USA). Ampli W cation was arrested after25 cycles (94°C £ 30s, 56°C £ 30s, and 72°C £ 30s).The samples were subjected to agarose gel electrophore-sis (1%) and visualized with ethidium bromide. Results Expression of luciferase after tail vein injection of DNAcomplexes Following tail vein injection of 100  g of the pEGFP-Luc vector as naked DNA or complexed to liposomes,polyethylenimine (PEI) or galactose conjugated PEI(PEI-Gal), the activity of luciferase was determined inliver, lung, kidney, spleen, and brain in wild type(C57BL/6) mice (Fig.1). The highest level of luciferaseexpression after 24h was found in lung using the lipo-somes with lower levels in the other tissues. Using PEI orPEI-Gal, the highest luciferase activity was also found inlung with barely detectable levels in the other tissues.Injection of naked DNA resulted in very low or unde-tectable levels of luciferase expression in all tissues. Expression of luciferase after portal vein injection of DNAcomplexes To improve transgene expression in liver, 100  g of pEGFPLuc, naked or complexed to liposomes, PEI orPEI-Gal, were injected into the portal vein withtransient blood X ow blockage. At 24h after injection,the highest luciferase expression was found in liverusing naked DNA with lower expression in the othertissues (Fig.2). Using PEI and PEI-Gal, higher lucifer-ase expression levels in liver were also observed as com-  A. Johansson et al. / Molecular Genetics and Metabolism 82 (2004) 20–26  23 pared with tail vein injection. Using liposomes, lowluciferase expression was found in all tissues and areasof extensive hepatic necrosis were found at autopsy inthese animals. DNA dose dependence on luciferase expression The e V  ect of the amount of naked pEGFPLuc (25– 400  g) on luciferase expression was studied in wild typemice 24h after portal vein injection. The highest level of luciferase expression in liver was obtained after injectionof 100  g of pEGFPLuc (Fig.3). Expression of PBGD after portal vein injection of naked DNA The p-mPBGDhou vector was injected as nakedDNA (100  g) into the portal vein of the AIP mousemodel, as this gave the highest transgene expression inliver when using pEGFLuc. At di V  erent time-points (8– 144h) after injection, PBGD activity was measured inliver homogenates and compared with the endogenouslevel in AIP mice. No increased PBGD activity wasobserved (Fig.4). Only one treated AIP mouse showedincreased PBGD activity up to wild type after 8h, but Fig.1. Luciferase activity in wild type (C57BL/6) mice injected in tail vein with 100  g of the luciferase expression plasmid (pEGFPLuc), naked orcomplexed 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 24h after injection. Data are presented asmeans of two animals.Fig.2. Luciferase activity in wild type (C57BL/6) mice injected in portal vein, with transient blood X ow blockage, with 100  g of the luciferase expres-sion plasmid (pEGFPLuc), naked or complexed to cationic liposomes, polyethylenimine (PEI) or galactose conjugated PEI (PEI-Gal), in a volumeof500  L. The luciferase activity, expressed as RLU/mg protein, was analyzed in liver, lung, kidney, spleen, and brain tissue homogenate 24h afterinjection. Data are presented as means of two animals.  24 A. Johansson et al. / Molecular Genetics and Metabolism 82 (2004) 20–26  this could not be repeated (data not shown). As control,injection of physiological saline (500  L) resulted in nochange in the endogenous PBGD activity (Fig.4). Detection of plasmid DNA in liver DNA was extracted from liver tissue after portal veininjections of 100  g of naked p-mPBGDhou or pEGFP-Luc. The DNA was PCR-ampli W ed using primers spe-ci W c for the CMV promoter. The plasmid p-mPBGDhouwas detectable in liver as early as 8h after portal veininjection and was detectable for at least 144h (Fig.5).The hepatic presence of p-mPBGDhou at 24h afterinjection was compared with that of pEGFPLuc (Fig.5)from which the highest hepatic expression was found(Fig.2). Discussion Hepatic gene therapy o V  ers the possibility of prevent-ing or minimizing the severity of the acute neurologicattacks in patients with AIP. Recently, it was shown thatfunctional PBGD can be expressed in vitro [4] and cor-rect the biochemical defect in PBGD-de W cient cells byusing non-viral vectors [5]. Here we have extended the Fig.3. Dose–response analysis of luciferase activity versus amount of luciferase expression plasmid (pEGFPLuc) in wild type (C57BL/6) mice. Themice were injected in portal vein in combination with transient blood X ow blockage with increasing amounts of naked pEGFPLuc (25–400  g) in avolume of 500  L. The luciferase activity, expressed as RLU/mg protein, was analyzed in liver 24h after injection. Data are presented as means of twoanimals.Fig.4. PBGD activity in liver of the AIP mice after portal vein injection with transient blood X ow blockage of 100  g of naked p-mPBGDhou, codingfor the mouse housekeeping PBGD, in a volume of 500  L. The PBGD activity, expressed as pkat/g protein, was analyzed at di V  erent time-points(24–144h) after injection. As reference, the endogenous PBGD activity was measured in liver tissue from wild type and AIP mice. Data are presentedas means of two animals and means § SD of 6–12 animals.
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