BBRC Biochemical and Biophysical Research Communications 320 (2004) 998–1006 www.elsevier.com/locate/ybbrc
Prolonged liver-specific transgene expression by a non-primate lentiviral vector Reba Condiotti,a,* Michael A. Curran,b Garry P. Nolan,c Hilla Giladi,a Mali Ketzinel-Gilad,a Eitan Gross,d and Eithan Galuna a
Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem 91120, Israel b Division of Immunology, University of California Berkeley, Berkeley, CA 94720, USA c Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA d Department of Pediatric Surgery, Hadassah University Hospital, Jerusalem 91120, Israel Received 28 April 2004 Available online
Abstract Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hepatocytes; Gene therapy; Lentiviral vectors; Viral integration; Hydrodynamic injection; Metabolic liver disease
Stable expression of therapeutic genes in the liver may be beneficial for patients with a wide variety of hepatic and systemic diseases, including metabolic and infectious disorders, hemophilias, hypercholesterolemias, and lipid storage diseases. Development of lentiviral vectors has led to efficient delivery and long-term expression of transgenes to hepatocytes without observable toxicity and with no apparent requirement for cell proliferation [1,2]. However, HIV-based vectors still pose a variety of safety concerns related to pathogenicity and, therefore, may not be suitable in some settings. While these obstacles are presently being addressed, vector systems based on non-primate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and bovine immunodeficiency virus (BIV) may offer solutions to some * Corresponding author. Fax: +972-2-643-0982. E-mail address:
[email protected] (R. Condiotti).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.044
of these concerns [3,4]. FIV infects domestic cats and strays as well as large cats such as lions and pumas. It is a particularly attractive candidate for lentiviral vector development because there is no epidemiological evidence that FIV produces human infection or disease [5], although humans have been exposed to FIV by the same route as natural feline transmission, namely bites and scratches [6]. The ability of FIV-based vectors to infect terminally differentiated cells, such as macrophages, retinal cells, and brain cells [7–9], has made the virus a possibly safer delivery system for human gene therapy, specifically for delivery of transgenes to hepatocytes which are terminally differentiated, nondividing cells. Using second and third generation, three plasmid FIV vector systems, we established the suitability of FIV as liver-directed gene transfer agents. We determined, in vitro, the advantage of a liver-specific promoter in obtaining optimal gene expression and showed
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that the vector underwent integration into the host genome. In addition, we addressed the in vivo efficacy of systemic delivery of FIV-based vectors and found that they lead to long-term expression exclusively in the liver.
Materials and methods Plasmids Second-generation transfer vectors. Construction of pFLX-RSG, pFLX-CMG, pFLX-CPL (KS), and pFLX-5CL is described [3,10,11]. In pFLX-5CG, the lacZ from pFLX-5CL was replaced with GFP. FLX-hPGK was derived from pFLX-CPL (KS) by inserting the hPGK promoter and GFP taken from pRLLhPGK GFPsin18 [12] between the EcoRV–NotI sites. pFLX-hAAT/GFP was constructed by inserting the human alpha1 anti-trypsin (hAAT) promoter (kindly provided by Katherine Ponder, Washington University, St. Louis, MO) followed by GFP, between the SmaI and Acc651 sites of pFLX-CPL (KS). pFLX-hAAT/luc is a pFLXhAAT/GFP derivative carrying luc taken from pGL3 replacing the GFP gene. Third-generation transfer vector. The third-generation vector (pPanther) contains the HIV central polypurine tract (cPPT) and the woodchuck post-transcription regulatory element (WPRE) cloned 30 of the reporter gene (Curran et al., manuscript in preparation). This vector has a deletion in the U3 region of the 30 LTR (SIN vector). The pPan-hAAT/luc vector was built by insertion of MluI/XbaI hAAT promoter/luciferase fragment taken from pAH 57 into pPanther digested with MluI/NheI.
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FACS FACS analysis was performed with a FACSCalibur (Becton– Dickinson, San Jose, CA) using Cellquest software. Ten thousand events were acquired in list mode. Cells were stained with 1 lg/ml propidium idodide (Sigma) and dead cells were gated out. Mean fluorescence intensity (MFI) was determined on gated GFP+ cells. Alu PCR Genomic DNA was extracted from FLC4 and HepG2 cells (Puregene DNA Isolation Kit, Gentra Systems, Minneapolis, MN) three weeks after infection. An initial PCR was performed on 300 ng DNA using the Alu-specific oligonucleotide 50 -CAGTGCCAAGTG TTTGCTGACG-30 and the vector encoded R regions of the LTR, corresponding either to the sense strand: 50 -GGAGTCTCTTTGTTG AGGAC-30 or the anti-sense strand: 50 -GTCCTCAACAAAGAGAC TCCTC-30 . A second, “nested,” PCR was carried out with internal R region-specific oligonucleotides (sense: 50 -GACATGATGGCCCGGA TTCC or anti-sense: 50 -CTCCCTTGAGGCTCCCACAG-30 ). For in vivo studies, Alu PCR analysis was performed on genomic DNA extracted from murine livers (Puregene DNA Isolation Kit, Gentra Systems, Minneapolis, MN), 7 and 30 days following injection of viral particles. An initial PCR was performed on 300 ng DNA using one primer specific for ubiquitous repeats found in the mouse genome (B2 sense: 50 -GGCTGGTGAGATGGTTCAGT-30 ) [16] and a second primer from the GFP sequence (GFP antisense: 50 -AACTCCAGCAG GACCATG-30 ). A nested PCR was performed with 5 ll of the B2/ vector PCR product, using two internal primers in the vector genome derived from the GFP sequence (GFP sense: 50 -CCACCATGGTGAG CAAGGGCG-30 and GFP antisense: as in the first PCR) amplifying a fragment of 680 bps. Southern analysis
Cell lines 293T, HepG2 (ATCC HB-8065), Huh7 (Japanese Cancer Research Resources Bank-Cell, Tokyo), FLC4 [13], and Hep3B (ATCC HB8064) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/BRL, Grand Island, NY) containing 10% fetal calf serum, 1 mM L -glutamine, 100 U/ml penicillin, and 0.01 mg/ml streptomycin (Biological Industries, Kibbutz Beth Haemek, Israel). Production of viral particles Viral particles were produced by transfection of 293T cells with 8.4 lg transfer vector, 14 lg packaging vector pCPRDEnv [3], and 5.6 lg envelope vector pMDG (VSV-G) [14,15] per 10 cm plate using 75 ll of Fugene (Roche Diagnostics, Mannheim, Germany). Supernatants were collected 48 and 72 h post-transfection. Viral particles were concentrated 10-fold from pooled supernatants spun in an ultracentrifuge (Sorval Discovery 100) at 21,000 rpm for 90 min at 4 °C and resuspended in PBS. Viral titers were determined by FACS analysis of 293T infected with diluted viral supernatant which reproducibly yielded 3–6 106 TU/ml. Transducing units (TU) were determined according to the following formula: % GFP 100,000 cells/volume of supernatant. Infection of cells Hepatocellular carcinoma cell lines (1 105 cells/ well in a 12-well plate) (Nunc, Roskilde, Denmark) were infected with 2 ml of a supernatant (3–6 106 TU/ml) and 5 lg/ml polybrene (Sigma, St. Louis, MO). The same procedure was repeated 24 h later. Three days after second infection, cells were harvested for FACS analysis. For in vitro time kinetics studies, cells were passaged at 1:5 or 1:10 twice a week for up to 24 days.
Twenty-five microgram genomic DNA was digested overnight with PacI or KpnI and separated on a 1% agarose gel, transferred to a nylon membrane (GeneScreen Plus, NEN Life Science Products, Boston, MA), and probed with a 32 P-labeled GFP fragment. In vivo transduction and imaging All animal procedures were performed according to institutionapproved protocols (Hebrew University—-Hadassah Medical School Animal Facilities). DNA (15 lg/animal) or viral particles concentrated 10-fold from the original supernatant were injected into the tail vein of female Balb/c mice (8–10 weeks old) (Harlan, Israel) in a final volume of 0.2 or 1 ml PBS. The mice were imaged as previously described [17]. Liver enzymes ALT and AST levels in murine serum were measured on a Reflotron (Boehringer–Mannheim GmbH, Mannheim, W. Germany). Immunofluorescence Liver cryostat sections, 4–5 lm thick, were post-fixed on glass slides with 4% cold paraformaldehyde for 15 min at room temperature and stained as previously described [1], mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA), and observed with a Nikon Eclipse E600 microscope. Histology Formalin-fixed, paraffin-embedded liver samples were stained with hematoxylin and eosin (H&E).
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Results Comparison of internal promoters on transgene expression in hepatocellular carcinoma (HCC) cell lines To compare the strength of various internal promoters on transgene expression in hepatocyte lineages, HepG2, Huh7, FLC4, and Hep3B, cells were transduced with equivalent titers (3–6 106 TU/ml) of second-generation FIV-based vectors containing the GFP reporter gene driven by one of the following constitutive promoters: cytomegalovirus (CMV), Rous sarcoma virus (RSV), murine PGK (mPGK), or human PGK (hPGK) (Fig. 1A). The various internal promoters did not seem to affect viral titer and produced approximately the same transducing units per volume. Since the hAAT promoter is weakly expressed in 293T cells, we could not determine its titer and therefore, an equivalent volume of viral supernatant was used. FACS analysis was performed 3 days post-second infection and promoter strength was measured by assessing the level of mean fluorescence intensity (MFI) on gated GFP+ cells (Fig. 1B). In HepG2, Huh7, and Hep3B transduced cells, hPGK and hAAT were relatively strong promoters with no significant difference between them (p > 0:05).
These results suggest that the liver-specific hAAT promoter is an appropriate element for gene expression in hepatocyte cell lines. Time kinetics of gene expression and integration of FIV vectors into the host cell genome To assess the stability of gene expression over time, Huh7 and FLC4 were continuously passaged after infection and the percentage of GFP-expressing cells was determined by FACS analysis on days 4, 6, 10, 14, 17, and 24 post-infection (Fig. 2). In both cell lines, the percentage of GFP-positive cells was stable throughout the culture period and the percentage of GFP-positive cells correlated with the promoter as follows: hAAT > RSV > CMV. These results indicate that transgene expression levels are stable for at least 24 days. Promoter strength, as assessed by fluorescence intensity, tended to be stable in Huh7 and FLC4 throughout the duration of two independent experiments and no obvious difference was observed in MFI values between days 4 and 24, regardless of cell line or vector (Table 1). The one exception to this phenomenon was shown in FLC4 cells infected with FLX-CMV
Fig. 1. Comparison of promoter strength in cultured HCC cell lines transduced by FIV-based lentiviral vectors. (A) Schematic drawing of secondand third-generation constructs used as shown in their proviral form. Vectors carry an internal cassette for enhanced GFP or luc driven by one of the following promoters: CMV, RSV, mPGK, hPGK, or hAAT. In the third-generation vectors, the following cis-acting sequences are labeled: the cPPTCTS sequence from the HIV-1 pol gene, the viral LTRs with the U3 deletion, the Rev response element (RRE), and WPRE. (B) Promoter strength as measured by mean fluorescence intensity of human HCC-derived cell lines 3 days post-transduction (mean SE, n P 3).
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Fig. 2. Kinetics of GFP expression in human HCC cell lines following transduction by second-generation FIV-based vectors. Huh7 (A) and FLC4 (B) were transduced with VSV-G pseudotyped viral particles containing pFLX-CMV, pFLX-RSV, and pFLX-hAAT transfer vectors. At various times post-transduction, cells were harvested and analyzed for GFP expression by FACS analysis (mean SD, n ¼ 2).
Table 1 Time kinetics of mean fluorescence intensity Days post-infection
4 6 10 14 17 24 a
Huh7a
FLC4a
CMV
RSV
hAAT
CMV
RSV
hAAT
31 4 20 3 31 4 84 29 12 19 4
37 1 32 0 51 8 26 11 50 12 37 7
48 46 74 24 97 8 84 0 75 16 74 25
251 15 149 1 142 1 110 0 89 8 103 23
202 57 185 15 216 21 195 16 159 25 185 64
140 19 128 29 137 7 121 11 112 15 162 9
n ¼ 2.
wherein the level of expression was lower on day 24 than on day 4. On days 6, 10, and 14 post-Huh7 infection, MFI values of the hAAT promoter appeared to be higher than RSV and CMV. In order to investigate integration of provirus from FIV-based vectors into HCC cell lines, genomic DNA was extracted from HepG2 and FLC4 cells 19 days postinfection and PCR was performed using two consecutive PCRs (see Alu PCR in Materials and methods). As shown in Fig. 3A, DNA extracted from infected cells revealed a 100-bp product consistent with the primers used in the “nested” PCR whereas DNA extracted from uninfected cells lacked a signal. Integration of vector DNA into the host chromatin was further shown by Southern blot analysis (Fig. 3B). To distinguish between integrated and non-integrated vector genomes, the DNA was digested with an enzyme which does not cut within the vector (PacI) and a different enzyme which cuts once (KpnI) (Fig. 1A). The membrane was probed for the GFP sequence. The PacI digest yielded a smear on the gel in which all products were larger than the supercoiled plasmid (Fig. 3B, lanes 2 and 4 vs. lane 10). KpnI digest revealed a smear in which products were both smaller and larger than linear plasmid DNA cut with the same enzyme (Fig. 3B B, lanes 6 and 8 vs. lane 11) and suggests that the vector genome was randomly integrated into the host cell chromatin. Genomic DNA extracted from uninfected cells gave no signal (lanes 3, 5, 7, and 9). In order to address the possibility of the presence of episomal copies of the vector, a Southern blot analysis was performed on genomic DNA from uninfected HepG2 cells mixed with
plasmid DNA equal to 1 or 10 copies/genome. As seen in Fig. 3B, a single band was observed (lane 10 and 11) at a similar location as plasmid DNA cut with the same restriction enzyme (lane 13). This control experiment indicates that episomal DNA may be present in infected cells in small numbers which are undetectable in comparison to strong integrated viral DNA signals. Transduction of liver cells in vivo To test the efficacy of FIV-based vectors in vivo, gene expression was assessed in 8- to 10-week-old Balb/c mice receiving systemic delivery of second- and third-generation FIV transfer vectors (Fig. 1A), either naked DNA or concentrated viral particles. As shown in Fig. 4, luciferase expression was observed exclusively in the liver of mice injected with plasmid DNA (Fig. 4A, inset) and the same phenomenon was observed in mice treated with viral particles (Fig. 4B, inset). However, naked DNA led to peak expression 24 h post-injection and by day 14 following treatment, there was no detectable light emanating from the liver (Fig. 4A) whereas administration of viral particles led to stable levels of luciferase expression from 100 days to more than 7 months post-injection (Fig. 4B) (n ¼ 3). Expression resulting from injection of viral particles produced from second- and third-generation vectors was monitored up to 7 months post-transduction. Third-generation vectors consistently led to higher levels of transgene expression (data not shown). Therefore, the remaining experiments were performed using third-generation vectors.
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Fig. 3. Analysis of integration into the host cell genome. A “nested” PCR protocol was used to detect proviral DNA using a primer pair that binds the Alu sequences of the human genome and the R sequence of the FIV-LTR, followed by a PCR amplifying the R region that results in a 100-bp fragment. (A) Genomic DNA was extracted from HepG2 and FLC4 19 days post-transduction with FLX-hAAT and a 100-bp signal is exhibited from the “nested” PCR. (B) Southern blot analysis of liver DNA digested as indicated and probed for the GFP sequence in the vector (see Fig. 1A). Genomic DNA was extracted from FLC4 (infected cells, lanes 2 and 6; control cells, lanes 3 and 7) or HepG2 (infected cells, lanes 4 and 8; control cells, lanes 5 and 9). Genomic DNA extracted from uninfected HepG2 cells was mixed with 6 pg ( ¼ 1 copy/genome, lane 10) or 60 pg ( ¼ 10 copies/ genome, lane 11) of pFLX-hAAT/GFP and digested with KpnI. Plasmid DNA was digested with PacI which does not cut the vector (lane 12) or KpnI which cuts once (lane 13). A 1 kb DNA marker was loaded in lane 1.
Fig. 4. Long-term in vivo expression in the liver of mice transduced with second and third-generation FIV-based lentiviral vectors containing the liverspecific hAAT promoter. Balb/c mice were injected into the tail vein with (A) 1 ml PBS containing 15 lg DNA or (B) 1 ml containing 107 viral particles (n ¼ 3, mean SD). (C) Mice were injected with 0.2 or 1 ml PBS containing 107 viral particles (n ¼ 1 per injection volume). At various times following injection, light emission was monitored on a CCCD camera (see insets). (D) Transduction efficiency in the liver of mice 7 days post-injection with FIVbased lentiviral vectors containing GFP. Immunofluorescence labeling was performed with anti-GFP and FITC-conjugated secondary antibodies.
Systemic delivery of plasmid DNA in large volumes has been shown to enable high levels of gene expression in hepatocytes [18]. Presently, gene expression was
measured using this technique by injecting viral particles in large and small volumes. Higher levels of expression were achieved by rapidly injecting viral particles in a
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Fig. 5. Integration of vector sequences into murine liver. (A) Genomic DNA was extracted from livers of mice 7 (lanes 1 and 2) and 30 days (lanes 3 and 4) post-injection and a 680-bp signal is exhibited from the “nested” PCR as described in Materials and methods. A signal from genomic DNA extracted from control mice is absent (lanes 5 and 6). (B) Six months post-viral administration, the right lobe of the liver was removed and luc expression was measured 2, 7, 10, 15, and 23 days post-hepatectomy (C) CCCD camera images of luc expression in partially hepatectomized mouse.
volume of 1 ml than when the same viral titer was injected in a volume of 0.2 ml (Fig. 4C). In order to evaluate the efficiency of gene transfer, cryostat sections of livers were analyzed for transgene expression by fluorescence microscopy (Fig. 4D). The average number of cells displaying GFP fluorescence was counted in randomly chosen fields (left panel) and compared with the total number of cells present by DAPI nuclear staining (right panel). The frequency of GFP-positive liver cells was approximately 1% and the transduced cells exhibited typical morphology of hepatocytes, such as double-nucleated, round nuclei. In order to investigate possible in vivo vector integration, genomic DNA was extracted from livers of mice injected with viral vectors containing GFP and Southern blot analysis was performed using a restriction enzyme, PacI, which does not digest the vector. The GFP probe hybridized with multiple fragments of various lengths which were all larger than the supercoiled plasmid, indicating vector integration (data not shown). This result was confirmed by a PCR protocol with primers specific for ubiquitous repeats in the mouse genome and for the vector genome [19] followed by a second PCR using “nested” primers at the 50 or 30 end of the GFP gene. As seen in Fig. 5A, the expected 680-bp band was obtained in DNA extracted from treated mice (lanes 1–4) whereas no band was apparent in control mice receiving a PBS injection (lanes 5 and 6). An additional study was carried out as proof of principle of vector integration: 6 months following FIV vector administration, a partial hepatectomy was performed on one mouse by removing the right lobe of the liver and luc expression was measured in parallel with liver regeneration. As seen in Figs. 5B and C, gene expression decreased immediately following hepatectomy and subsequently increased with time reaching prehepatectomy levels 10 days post-surgery. The increase in transgene expression in parallel with liver regeneration indicates vector integration.
Fig. 6. Liver toxicity analysis. Hepatocellular cytotoxicity was analyzed by measuring serum AST and ALT levels in serum derived from mice 1, 7, and 30 days post-injection with viral particles, PBS, or untreated controls (mean SD, n P 3).
Liver damage resulting from the high volume injection or toxicity due to the transgene was assessed. One day post-infection, both AST and ALT levels were slightly elevated in both virus- and PBS-injected mice as compared to controls indicating mild damage (Fig. 6). Seven days following injection, liver enzyme levels decreased to those of the controls and remained stable 30 days post-administration. Tissue pathology reports supported these findings, namely, that mild liver damage due to a hydrodynamics injection is transient and expression of GFP does not indicate toxicity. Immune response to specific FIV viral proteins was not assessed.
Discussion FIV displays many features that render it desirable as a vector system. Like all retroviruses, genes carried by lentiviral vectors are stably integrated into the target cell genome. Most importantly, however, the ability of lentiviral vectors to transduce non-dividing cells, such as hepatocytes, is particularly attractive [3]. In addition to the benefits of human lentiviral vectors, FIV-based vectors could offer safety advantages. In contrast to
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HIV-based vectors, an FIV vector system permits in vivo testing of the safety and properties of a lentivirus vector in animals routinely susceptible to disease caused by the parental lentivirus [20]. In this study, we investigated the use of FIV-based lentiviral vectors for liver-targeted gene delivery. Vectors containing a variety of internal promoters driving the gene for GFP efficiently transduced HCC and led to long-term stable expression. In three out of four cell lines transduced with approximately equivalent viral titers, hAAT, a liver-specific promoter was at least as strong as other promoters. The efficacy of liver-specific promoters for high-level sustained reporter gene expression has been previously demonstrated in cultured HCC cell lines, primary hepatocytes, and transgenic mice [21,22]. A possible explanation is that tissue-specific promoters have transcriptional advantage over viral or other ubiquitous promoters which may express poorly in hepatocytes and could undergo silencing. As seen in the time kinetics study (Fig. 2), there is no evidence of hAAT promoter silencing and therefore can be advantageous for long-term expression. An additional advantage of using a liver-specific promoter is its ability to target a particular tissue [1]. In the present study, transgene expression was found to be stable over time, in vitro, and we assessed the possibility of vector integration into the host cell genome to account for this phenomenon. PCR analyses using human Alu sequences and Southern blot analysis suggest that transduced HCC cell lines did, indeed, harbor proviral sequences, 19 days post-transduction. Ikeda et al. [23] showed that FIV DNA is detectable in high-molecular-weight DNA for extended periods of time in a human cell line and concluded that FIV provirus undergoes integration. Pfeifer et al. [2] extended these findings in vivo and found that PCR analysis of proviral DNA showed vector integration in the liver 3 weeks after injection of the HIV-based lentiviral vectors. Our studies indicate that FIV-based vectors are similar to HIV vectors in their ability to integrate into the host cell genome. The location(s) of FIV vector integration is still unknown. This issue must be addressed since the risk of insertional mutagenesis of retroviral vectors has recently been a cause of great concern [24]. Some lessons may be learned from the work of Schroder et al. [25] who found that HIV-1 integration in the human genome favors active genes and local hotspots and therefore may not be a totally random occurrence. It has also been shown that HIV-based vectors can undergo integration in multiple sites in NOD/SCID repopulating cells which may increase the risk of insertional mutagenesis [26]. Whether we can apply this valuable information to FIV-based vectors in the murine genome remains to be seen. The duration of gene expression was compared between mice receiving high volume administration of
plasmid DNA and those receiving VSV-G pseudotyped viral particles. This recent “hydrodynamics” approach for DNA delivery to hepatocytes causes high-pressure conditions which enables the extravasation of plasmid DNA, perhaps through disruption of tight junctions or an increase in sinusoid fenestrae size. We and others found that this technique does, indeed, lead to gene expression targeted to the liver, although the expression is short-lived in those receiving naked DNA [18,27,28] possibly due to interference from the CMV promoter located in the 50 LTR. Herein, we adapted this technique by injecting viral particles under high-pressure [29]. This led to a similar liver-directed transduction, but was longlasting, suggesting vector integration into the cellular DNA. The likelihood of in vivo integration was supported by Southern blot analysis and Alu PCR. Partial hepatectomy leads to liver regeneration through cell division [30]. As transduced cells divide, integrated transgene expression is predicted to rise in parallel. As expected from Southern blot and Alu PCR results, luciferase expression in the livers of transduced mice increased with time following partial hepatectomy. A steady enhancement of transgene expression was observed up to 100 days following transduction and this phenomenon has not yet been addressed. It was previously shown that the half-life of a murine hepatocyte is approximately 180 days [31]. One possibility is that immature transduced liver cells divide causing an increase in transgene expression while older hepatocytes are dying. From day 100 onward, transgene expression was stable and no significant difference was found in luciferase activity between day 100 and day 210 posttransduction (n ¼ 3). In vivo administration of viral particles produced from third-generation vectors containing cis-acting elements led to higher expression than second-generation vectors. One element which was restored in the modified vector was the cPPT-CTS sequence from the gene, pol, of HIV [32] which is known to enhance nuclear translocation of the vector genome and increase the maximal frequency of transduction and transgene expression in most target cells tested, including primary hepatocytes [1]. The WPRE is the second element included in the third-generation vector. In the nucleus, the presence of the WPRE stimulates mRNA poly(A) tail length and facilitates other aspects of RNA processing including RNA export. In the cytoplasm, the poly(A) tails of messages containing the WPRE maintain the length observed in the nucleus. These activities lead to RNA stability and therefore stimulate transgene expression [33]. In addition to the two aforementioned modifications, the third-generation vector contains a deletion in the U3 region of the LTR. The U3 region in the 30 LTR of an infectious retroviral RNA serves as a template for the formation of both U3 regions during replication of retroviruses. Therefore, a deletion in the U3 enhancer
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region of the 30 LTR will be transferred to both LTRs in the provirus. This reduces the transcriptional activity of the LTR promoter by as much as 85–90% but exerts minimal effects upon an internally placed promoter [34]. This deletion not only makes the vectors safer but also prevents promoter competition. The combination of modifications made in the third-generation vector successfully upregulated transgene expression in vivo. In summary, our data indicate that: (1) FIV-vectormediated gene transfer to the liver may offer continuous transgene expression due to its ability to integrate into the host cell genome; (2) intravenous hydrodynamic delivery of third-generation FIV-based vectors predominantly transduces the liver and provides a persistent supply of a transgene product in a murine model; and (3) the use of a tissue-specific promoter appears to be advisable if sustained expression in a particular target tissue is indicated. These results are encouraging and illustrate that gene transfer to hepatocytes with FIVbased vectors may have potential curative value for certain inherited diseases.
Acknowledgments We thank Evelyne Zeira (Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital) and Meir Ohana (Liver Unit, Hadassah University Hospital) for their assistance with the animal studies. This study was supported by a grant from the Israeli Ministry of Science, an infrastructure supportive grant from Hadassah Hospital Funds, and the Grinspoon and Blum Foundations.
[9]
[10] [11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
References [1] A. Follenzi, G. Sabatino, A. Lombardo, C. Boccaccio, L. Naldini, Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors, Hum. Gene. Ther. 13 (2002) 243–260. [2] A. Pfeifer, T. Kessler, M. Yang, E. Baranov, N. Kootstra, D.A. Cheresh, R.M. Hoffman, I.M. Verma, Transduction of liver cells by lentiviral vectors: analysis in living animals by fluorescence imaging, Mol. Ther. 3 (2001) 319–322. [3] M.A. Curran, S.M. Kaiser, P.L. Achacoso, G.P. Nolan, Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors, Mol. Ther. 1 (2000) 31–38. [4] M.A. Curran, G.P. Nolan, Recombinant feline immunodeficiency virus vectors: preparation and use, Methods Mol. Med. 69 (2002) 335–350. [5] N. Nowotny, A. Uthman, O.A. Haas, A. Borkhardt, K. Lechner, H.F. Egberink, K. Mostl, M.C. Horzinek, Is it possible to catch leukemia from a cat?, Lancet 346 (1995) 252–253. [6] N.C. Pederson, The feline immunodeficiency virus, in: J.A. Levy (Ed.), The Retroviridae, Plenum Press, New York, 1993, pp. 181– 228. [7] J. Johnston, C. Power, Productive infection of human peripheral blood mononuclear cells by feline immunodeficiency virus: implications for vector development, J. Virol. 73 (1999) 2491– 2498. [8] A.J. Lotery, T.A. Derksen, S.R. Russell, R.F. Mullins, S. Sauter, L.M. Affatigato, E.M. Stone, B.L. Davidson, Gene transfer to the
[19] [20]
[21]
[22]
[23]
[24]
1005
nonhuman primate retina with recombinant feline immunodeficiency virus vectors, Hum. Gene Ther. 13 (2002) 689–696. A.I. Brooks, C.S. Stein, S.M. Hughes, J. Heth, P.M. McCray Jr., S.L. Sauter, J.C. Johnston, D.A. Cory-Slechta, H.J. Federoff, B.L. Davidson, Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus-based vectors, Proc. Natl. Acad. Sci. USA 99 (2002) 6216–6221. M.A. Curran, G.P. Nolan, Non-primate lentiviral vectors, Curr. Top. Microbiol. Immunol. 261 (2002) 75–105. M.A. Curran, G.P. Nolan, Nonprimate lentiviral vectors, Curr. Top. Microbiol. Immunol. 261 (2002) 75–105. R. Zufferey, T. Dull, R.J. Mandel, A. Bukovsky, D. Quiroz, L. Naldini, D. Trono, Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery, J. Virol. 72 (1998) 9873– 9880. H. Sujino, S. Nagamori, K. Fujise, S. Hasumura, S. Homma, T. Matsuura, K. Shimizu, H. Kameda, A. Tanaka, The study of morphological changes of human cultured hepatoma cells at elevated temperature, J. Clin. Electron Microsc. 19 (1986) 5–6. L. Naldini, U. Blomer, P. Gallay, D. Ory, R. Mulligan, F.H. Gage, I.M. Verma, D. Trono, In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science 272 (1996) 263–267. D.S. Ory, B.A. Neugeboren, R.C. Mulligan, A stable humanderived packaging cell line for production of high titer retrovirus/ vesicular stomatitis virus G pseudotypes, Proc. Natl. Acad. Sci. USA 93 (1996) 11400–11406. K.L. Bennett, R.E. Hill, D.F. Pietras, M. Woodworth-Gutai, C. Kane-Haas, J.M. Houston, J.K. Heath, N.D. Hastie, Most highly repeated dispersed DNA families in the mouse genome, Mol. Cell. Biol. 4 (1984) 1561–1571. A. Honigman, E. Zeira, P. Ohana, R. Abramovitz, E. Tavor, I. Bar, Y. Zilberman, R. Rabinovsky, D. Gazit, A. Joseph, A. Panet, E. Shai, A. Palmon, M. Laster, E. Galun, Imaging transgene expression in live animals, Mol. Ther. 4 (2001) 239–249. G. Zhang, V. Budker, J.A. Wolff, High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA, Hum. Gene Ther. 10 (1999) 1735–1737. S.L. Butler, M.S. Hansen, F.D. Bushman, A quantitative assay for HIV DNA integration in vivo, Nat. Med. 7 (2001) 631–634. E.M. Poeschla, F. Wong-Staal, D.J. Looney, Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors, Nat. Med. 4 (1998) 354–357. K.P. Ponder, R.P. Dunbar, D.R. Wilson, G.J. Darlington, S.L. Woo, Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection, Hum. Gene Ther. 2 (1991) 41–52. C.A. Pinkert, D.M. Ornitz, R.L. Brinster, R.D. Palmiter, An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice, Genes Dev. 1 (1987) 268–276. Y. Ikeda, K. Tomonaga, Y. Kawaguchi, M. Kohmoto, Y. Inoshima, Y. Tohya, T. Miyazawa, C. Kai, T. Mikami, Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells, J. Gen. Virol. 77 (8) (1996) 1623–1630. S. Hacein-Bey-Abina, C. Von Kalle, M. Schmidt, M.P. McCormack, N. Wulffraat, P. Leboulch, A. Lim, C.S. Osborne, R. Pawliuk, E. Morillon, R. Sorensen, A. Forster, P. Fraser, J.I. Cohen, G. de Saint Basile, I. Alexander, U. Wintergerst, T. Frebourg, A. Aurias, D. Stoppa-Lyonnet, S. Romana, I. RadfordWeiss, F. Gross, F. Valensi, E. Delabesse, E. Macintyre, F. Sigaux, J. Soulier, L.E. Leiva, M. Wissler, C. Prinz, T.H. Rabbitts, F. Le Deist, A. Fischer, M. Cavazzana-Calvo, LMO2associated clonal T cell proliferation in two patients after gene therapy for SCID-X1, Science 302 (2003) 415–419.
1006
R. Condiotti et al. / Biochemical and Biophysical Research Communications 320 (2004) 998–1006
[25] A.R. Schroder, P. Shinn, H. Chen, C. Berry, J.R. Ecker, F. Bushman, HIV-1 integration in the human genome favors active genes and local hotspots, Cell 110 (2002) 521–529. [26] N.B. Woods, A. Muessig, M. Schmidt, J. Flygare, K. Olsson, P. Salmon, D. Trono, C. von Kalle, S. Karlsson, Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis, Blood 101 (2003) 1284–1289. [27] H. Herweijer, G. Zhang, V.M. Subbotin, V. Budker, P. Williams, J.A. Wolff, Time course of gene expression after plasmid DNA gene transfer to the liver, J. Gene Med. 3 (2001) 280–291. [28] F. Liu, Y. Song, D. Liu, Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA, Gene Ther. 6 (1999) 1258–1266. [29] G. Zhang, Y.K. Song, D. Liu, Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous
[30] [31] [32]
[33]
[34]
administration of plasmid DNA using a hydrodynamics-based procedure, Gene Ther. 7 (2000) 1344–1349. N. Fausto, Liver regeneration, J. Hepatol. 32 (2000) 19–31. C.J. Steer, Liver regeneration, Faseb J. 9 (1995) 1396– 1400. V. Zennou, C. Serguera, C. Sarkis, P. Colin, E. Perret, J. Mallet, P. Charneau, The HIV-1 DNA flap stimulates HIV vectormediated cell transduction in the brain, Nat. Biotechnol. 19 (2001) 446–450. E.A. Choi, J. Loeb, T.J. Hope, Mechanism of the woodchuck hepatitis virus post-transcriptional regulatory element WPRE, Mol. Ther. 3 (2001) S397. D.G. Hafenrichter, K.P. Ponder, S.D. Rettinger, S.C. Kennedy, X. Wu, R.S. Saylors, M.W. Flye, Liver-directed gene therapy: evaluation of liver specific promoter elements, J. Surg. Res. 56 (1994) 510–517.
