Gene Therapy (1999) 6, 114–120 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line MA Zern1,2, I Ozaki1,3, L Duan1, R Pomerantz1, S-L Liu1, and DS Strayer2 Departments of 1Medicine and 2Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA, USA Alpha 1-antitrypsin (␣1AT) deficiency disease is one of the ribozyme cleavage. Ribozymes were effective in inhibiting more common hereditary disorders that affects the liver ␣1AT expression in a human hepatoma cell line using a and lung. The liver disease of ␣1AT deficiency is generally newly developed simian virus (SV40) vector system. In thought to be caused by the accumulation of an abnormal addition, the hepatoma cell line was stably transduced with ␣1AT protein in hepatocytes, whereas the lung disease is a modified ␣1AT cDNA that was capable of producing wild- thought to be due to a relative lack of the normal protein type ␣1AT protein, but was not cleaved by the ribozyme in the circulation. Therefore, one possible approach to pre- that decreased endogenous ␣1AT expression. These vent and treat ␣1AT disease is to both inhibit the results suggest that ribozymes can be employed for the expression of the mutated ␣1AT gene, and to provide a specific inhibition for an abnormal ␣1AT gene product, the means of synthesizing the normal protein. To do this, we first step in designing a gene therapy for the disease. The designed specific hammerhead ribozymes that were cap- findings also suggest that the novel SV40-derived vector able of cleaving the ␣1AT mRNA at specific sites, and con- may represent a fundamental improvement in the gene structed a modified ␣1AT cDNA not susceptible to therapeutic armarmentarium. Keywords: ribozymes; SV40; gene therapy; ␣1-antitrypsin deficiency Introduction Lys substitution at amino acid 342. This mutation is thought to cause the variant protein to aggregate in the ␣ Alpha 1-antitrypsin ( 1AT) deficiency, one of the more rough endoplasmic reticulum of the liver cells.4,6 common lethal hereditary disorders in Caucasians of Eur- Although several studies have focused on the delivery opean descent, is characterized by reducing serum levels of the normal ␣1AT gene into hepatocytes or airway cells ␣ of 1AT, a 52-kDa glycoprotein that functions as an anti- to restore normal ␣1AT production and to protect lung 1 protease. The deficiency state is caused by mutations of tissue,7–9 these approaches do not affect the liver disease. ␣ 2 the 1AT gene, a pleiomorphic, 12.2-kb 7-exon gene. It would appear that the best way to treat ␣1AT ␣ ␮ 3 Normal 1AT serum levels are 20–53 m; various combi- deficiency disease is to reduce the production of the ␣ nations of at least 17 different mutations of the 1AT endogenous mutant form of ␣1AT protein and to increase ␣ Ͻ ␮ gene are associated with an 1AT level 11 m and sig- the synthesis of the normal protein. The reduction of 4 nificant risk for developing emphysema. A subset of ␣1AT expression can be achieved by several strategies, 5 mutations is associated with hepatitis and cirrhosis. such as antisense, gene-specific ribozymes, ␣1AT tran- These latter mutations all involve the production of scription factor specific-inhibitors, or intracellular abnormal proteins: they do not include null mutations. expression of antibody to the mutant form of the ␣1AT The pathogenesis of the liver disease is thought to be due protein. ␣ to the accumulation of an abnormal 1AT protein in Recently, ribozymes have been targeted to a wide var- hepatocytes, and is associated with the finding that cer- iety of substrates and tested in biological systems to ␣ tain mutations of the 1AT gene cause derangement in achieve the inhibition of cellular gene expression or viral the protein’s intracellular processing and defects in the replication.10–15 Ribozymes have an advantage in the ther- protein’s excretion, commonly associated with liver apy of chronic disease since they are not degraded when 6 injury. The molecular defect in the protease inhibitor the target RNA is cleaved, and therefore, theoretically (Pi)Z allele, the allele most commonly associated with they need not be produced at higher levels than the target liver injury, is a G to A transition resulting in a Glu to transcript. By using a hammerhead ribozyme, we should be able to target the mutant form of ␣1AT mRNA by using ␣1AT guide sequences attached to the ribozyme Correspondence: MA Zern, Jefferson Medical College, 1025 Walnut catalytic core sequence. Furthermore, if we change the Street, Room 901, Philadelphia, PA 19107, USA corresponding cloned wild-type ␣1AT cDNA GUC and 3Present address: Department of Internal Medicine, Saga Medical School, Saga, Japan guide region nucleotide sequences while maintaining the The first two authors contributed equally to this work amino acid sequence, this modified ‘wild-type’ ␣1AT Received 7 November 1997; accepted 13 August 1998 mRNA should be resistant to the ribozyme. SV40-transduced ribozymes inhibit ␣1-antitrypsin MA Zern et al 115 A major issue in designing adequate gene therapeutic approaches to diseases is the availability of an effective vector system that will both deliver the gene of interest to the target organ and allow for its adequate expression. One of our laboratories has recently engineered the simain virus (SV)40 so that it has become an effective transfer vector system.16,17 In the present study, we report the transduction of an effective ␣1AT ribozyme in this SV40 vector system to inhibit the expression of the ␣1AT gene in a human hepatoma-derived cell line. At the same time, we have constructed a modified ␣1AT cDNA that is capable of producing the normal ␣1AT protein, yet its RNA is resistant to ribozyme cleavage. Results To study the effects of ribozymes on the expression of ␣1AT in a human hepatoma-derived cell line, we trans- duced PLC/PRF/5 cells that actively produce ␣1AT with either retroviral or SV40 recombinant viruses. Both poly- Figure 1 Representative Northern blot of RNA extracted from an unselec- ted population of PLC/PRF/5 cells that were mock-infected or infected 48 h merase II and III promoters were employed to drive ␣ expression of the ribozymes. previously with an SV40-derived vector containing an 1AT ribozyme 18 driven by either the SV40 early promoter (SVP.AT589) or by a tRNA In one series of studies, retroviral vectors were used promoter [SV(⌬)AT204] or [SV(⌬).tAT589T] at an MOI of 10. The RNA to transfer ribozymes. RNA was extracted from a pooled was electrophoresed, then hybridized with ␣1AT or GAPDH probes, as population of cells that had been tranduced with the described in Materials and methods. retroviral vectors and selected with G418 for 3 weeks. Those studies indicated that whereas ribozyme expression driven by a tRNA promoter decreased ␣1AT mRNA expression by as much as 85%, no such effect on ␣1AT mRNA expression was found when the CMV pro- moter was used to drive ribozyme expression. The SV40 vector system was somewhat more effective in that ribozymes driven by a polymerase II promoter were effective when transduced with the new vector system. PLC/PRF/5 cells were transduced using three different SV40 constructs, each containing either AT204 or 589 ribozyme. One construct employed the AT204 ribozyme driven by the tRNA promoter, one used the AT589 ribozyme under the control of the SV40 early promoter, and the third used the AT589 ribozyme with the tRNA promoter. No selection was applied to these cells. Forty- eight hours following transduction, RNA was isolated from the unselected populations of cells and assayed by Northern blot hybridization analysis. ␣1AT mRNA levels were significantly decreased in cells infected with the recombinant viruses in which ribozyme expression were driven by either the SV40 early promoter or the tRNA promoter, whereas there was no difference in GAPDH expression (Figure 1). Further studies were done with ribozyme AT589 con- Figure 2 Northern blot hybridization analysis of ␣1AT ribozymes employing an SV-40 vector system. PLC/PRF/5 cells were infected in cul- structs because they generated consistently high ⌬ ⌬ ribozyme activity. Figure 2 is a series of representative ture, with SV( )CAT, SVP.AT589, or SV( ).tAT589 at MOI of approxi- mately 100, or they were mock-infected. Cells were infected and RNA Northern blots of RNA isolated from an unselected popu- extracted as in Materials and methods. SV(⌬)CAT represents a control lation of cells 48 h following transduction with a control for transduction. construct, SVCAT, or with vector constructs containing 589AT ribozymes. The blots demonstrate the effective- ness of the ribozymes and their selective and specific despite the cells having a doubling time of 3 days. This properties. suggests that the construct was integrated, although no In addition, we employed the AT589 construct in proof of integration was demonstrated. Another alterna- experiments of longer duration to demonstrate the effec- tive explanation is that the high MOI employed allows tiveness of the construct over time. The PLC/PRF/5 cells for episomal expression in daughter cells. were transduced with the SV(⌬).tAT589 construct, then To compensate for the endogenous ␣1AT expression RNA was extracted at days 2, 7 and 14 following trans- being reduced by the AT589 ribozyme, we modified the duction. Figure 3 demonstrates that the ribozyme effec- nucleotide sequences of the target site of AT589 in the tively cleaved the ␣1AT mRNA after 2 weeks of infection full-length ␣1AT cDNA, without changing amino acid SV40-transduced ribozymes inhibit ␣1-antitrypsin MA Zern et al 116 Figure 3 Northern blot hybridization analysis of ␣1AT and GAPDH mRNA in a PLC/PRF/5 cell population mock-infected or infected with an SV40-derived vector containing the AT589 ribozyme.