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Retroviral-Mediated Gene Transfer and Expression of Human Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8146-8150, November 1988 Genetics Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes HONG PENG*, DONNA ARMENTANO*t, LESLIE MACKENZIE-GRAHAM*t, RONG-FONG SHEN*t, GRETCHEN DARLINGTONt, FRED D. LEDLEY*t, AND SAVIo L. C. WOO*t Departments of *Cell Biology and tPathology and Institute for Molecular Genetics, tHoward Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030 Communicated by Earl W. Davie, July 27, 1988 (received for review April 20, 1988) ABSTRACT Genetic therapy for phenylketonuria (severe infection of primary mouse hepatocytes with recombinant phenylalanine hydroxylase deficiency) may require introduc- retroviruses containing a neomycin-resistance marker gene. tion of a normal phenylalanine hydroxylase gene into hepatic Primary hepatocytes infected with the recombinant retrovi- cells of patients. We report development of a recombinant ruses were transformed to neomycin resistance and continue retrovirus based on the N2 vector for gene transfer and ex- to express liver-specific function (9). pression of human phenylalanine hydroxylase cDNA in primary We now report construction of a recombinant retrovirus mouse hepatocytes. This construct contains an internal pro- containing the human PAH cDNA under the control of moter ofthe human a1-antitrypsin gene driving transcription of transcriptional regulatory elements of the human a1- the phenylalanine hydroxylase cDNA. Primary mouse hepa- antitrypsin (a1AT) gene. These promoter elements have been tocytes were isolated from newborn mice, infected with the shown to direct high-level tissue-specific expression of a recombinant virus, and selected for expression ofthe neomycin- chloramphenicol acetyltransferase marker gene in cultured resistance gene. Hepatocytes transformed with the recombinant hepatoma cells (10) and the liver oftransgenic mice (R.-F.S., virus contained high levels ofhuman phenylalanine hydroxylase unpublished results). The recombinant retrovirus was used to mRNA transcripts originating from the retroviral and internal infect primary mouse hepatocytes and shown to direct promoters. These results demonstrate that the transcriptional high-level expression of human PAH mRNA. regulatory elements of the a1-antitrypsin gene retain their tis- sue-specific function in the recombinant provirus and establish MATERIALS AND METHODS a method for efficient transfer and high-level expression of Construction of pNAS-hPAH. The Sma I-EcoRI fragment human phenylalanine hydroxylase in primary hepatocytes. from the human PAH cDNA phPAH247 (bases 213-2453) (11) was cloned into the HindIII site of a pAlOCAT2-based Phenylalanine hydroxylase (PAH, EC 1.14.16.1) is a tissue- construct (10, 12), which contains the TATA box and specific enzyme that in humans is expressed only in the liver initiation site from the simian virus 40 (SV40) early promoter (1). PAH converts phenylalanine to tyrosine by using tetrahy- plus a 700-base-pair (bp) Ava I-BamHI fragment (-730 to drobiopterin as a cofactor (2). Deficiency of PAH results in - 30) from human ajAT (Fig. 1). This fragment of the ajAT the disease phenylketonuria (PKU), in which failure of gene has been shown to contain cis-acting regulatory ele- normal phenylalanine catabolism leads to accumulation of ments for tissue-specific expression in hepatic cells (10). phenylalanine and abnormal metabolites of phenylalanine. A 2-kb fragment containing the a1AT/SV40 hybrid pro- This, in turn, causes a phenotype of severe mental retarda- moter and the PAH cDNA [devoid of the poly(A) site] was tion (3-5). Treatment for PKU currently consists of limiting recovered by Sal I-Hpa I digestion and blunt-end cloned into phenylalanine intake with a protein-restricted, elemental the Xho I site of retroviral vector N2, which contains formula diet (3-5). retroviral LTRs, an extended packaging sequence, and the Somatic gene therapy for PKU may be considered, in Tn5 neomycin-resistance gene (13). This construct is desig- which a recombinant phenylalanine hydroxylase gene is nated pNAS-hPAH (Fig. 1). introduced into cells of patients with PKU in order to restore Cells and Medium. The ecotropic and amphotropic virus PAH enzymatic function. Preliminary studies have demon- packaging cell lines 4i2 (14) and PA317 (15) were kindly strated that the PAH apoenzyme can be reconstituted in provided by Richard Mulligan and Dusty Miller, respec- established cell lines by DNA-mediated (6) or viral-mediated tively. Cells were cultured in Dulbecco's modified Eagle's (7) gene transfer of a recombinant human PAH cDNA. These medium (DMEM) or 3:1 minimal essential medium studies illustrated that PAH holoenzyme activity in trans- (MEM)/Waymouth's MAB medium with 10% fetal calf formed cells is observed only when the reduced biopterin serum in 10% CO2. Media and fetal calf serum were pur- cofactor is available. Hepatoma cells are able to synthesize chased from Hazelton Research Products (Lenexa, KS). tetrahydrobiopterin from GTP and will support PAH activity Production of Amphotropic Recombinant Retrovirus. The (7). Fibroblast-like cells (NIH 3T3) are unable to synthesize plasmid pNAS-hPAH was transfected into the ecotropic biopterin and require exogenous administration of biopterin packaging cell line q,2 by calcium phosphate precipitation as in order to support PAH enzymatic activity (8). Therefore described (15, 16). The recombinant virus produced by tran- somatic gene therapy for PKU may require restoration of sient expression ofthe transfected 42 cells was harvested after PAH gene function in the liver where the biopterin cofactor 48 hr from conditioned medium by filtration through 0.45-.um is normally produced. Millipore filters. The amphotropic packaging cell line PA317 Retroviral-mediated gene transfer has been shown to be a was infected by exposure to virus for 2 hr in the presence of powerful method of introducing genes into a variety of 8 ,ug of Polybrene per ml and selected for expression of primary cells. We have previously reported the successful neomycin-resistance gene with G418 (500 ,ug/ml) at 24 hr. The publication costs of this article were defrayed in part by page charge Abbreviations: ajAT, a1-antitrypsin; PAH, phenylalanine hydroxy- payment. This article must therefore be hereby marked "advertisement" lase; LTR, long terminal repeat; SV40, simian virus 40; PKU, in accordance with 18 U.S.C. §1734 solely to indicate this fact. phenylketonuria. 8146 Downloaded by guest on October 2, 2021 Genetics: Peng et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8147 SV40 5'al-AT Early Promoter PAH cDNA (2034) (-730) (-30) (1 28) (51 71 ) (213) Hpa I (2453) Ava I B mHI Sph I Hind III Sma I EcoRI lZ ZI L Cap '1 IATGJ / [TAAJ [AATAAA] SV40 l TATAA c a1AtroICap *AH cDN_ -ME I- I (-730) (-30) (Hpa I) LTR NEO LTR - pNASPAH I1I I I Sac I (Xhol) Sac. I Viron RNA 6.0 kb Spliced RNA 5.1 kb _________ PAH mRNA 2.8 kb FIG. 1. Schematic of NAS-hPAH vector and RNA transcripts from the vector. The NAS-hPAH vector contains (i) a 700-bp fragment (- 730 to - 30) from the human ajAT gene representing cis-acting regulatory elements for tissue-specific promoter function (10); (ii) the 198-bp fragment ofSph I (128)-Hindlll (5171) from SV40 representing the enhancerless early promoter and cap site (13); and (iii) bases 210-2034 from the human PAH cDNA representing the open reading frame and devoid of most 5' and 3' untranslated sequences (11). This minigene is cloned into the Xho I site of the retroviral vector N2, which contains the Tn5 neomycin-resistance gene (13). Transcription from the 5' long terminal repeat (LTR) promoter in the provirus will result in a full-length proviral transcript of6.0 kilobases (kb) as well as a 5.1-kb mRNA representing a spliced mRNA product from an internal cryptic splice site (13). Transcription from the internal ajAT/SV40 promoter will result in a 2.8-kb mRNA. G418-resistant colonies were subcloned and virus production were isolated and analyzed for virus titer. One cell line was titered by infection ofrat 208 F cells as described (15, 17). (NAS-hPAH25) had a titer of 2 x 105 colony-forming units Culture and Infection ofPrimary Hepatocytes. Hepatocytes (cfu)/ml. Ten had virus titers of -0-104 cfu/ml. The rest were isolated from 3- to 7-day-old C57B/6 mice as described had no detectable virus at 10'- dilution. A parallel experi- (9). Cells were plated on Primaria tissue culture dishes and ment was performed with the N2 virus. Seven subclones infected with conditioned medium from virus-producing cells were isolated, with one clone (N2-4) yielding a virus-produc- diluted 1:1 with MEM/Waymouth's MAB, 10% fetal calf ing titer of 5 x 106 cfu/ml. serum, and Polybrene (final concentration, 4 gg/ml) for 12-hr DNA was prepared from the NAS-hPAH25 cell line. The periods at 12 and 14 hr. At 48 hr cells were placed in tyrosine- provirus was detectable as a 5.9-kb Sac I fragment hybrid- free MEM/Waymouth's MAB and SUM-3 hormonally de- izing to human PAH cDNA probe (Fig. 2, lane 2), which fined media (18). At % hr G418 (250 gg/ml) was added. Cells comigrated with the Sac I fragment of cloned NAS-hPAH were harvested 10 days after infection for analysis as de- plasmid DNA (Fig. 2, lane 1), indicating that there were no scribed (9). deletions in the proviral structure. No DNA hybridizing with Analytical Procedures. RNA was prepared by the hot the human PAH probe was detected in the N2-producing phenol method and RNA transfer blotting (Northern blotting) PA317 cell line under these conditions (Fig. 2, lane 3). was performed after formaldehyde/agarose gel electropho- Infection and Selection of Primary Mouse Hepatocytes. resis as described (7, 8). DNA was prepared as described and Primary mouse hepatocytes were infected with amphotropic analyzed by Southern blotting following digestion with the retroviruses N2-4 and NAS-hPAH25 and then selected with enzyme Sac I (6, 8).
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