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Blood, 1 April 2007, Vol. 109, No. 7, pp. 2797-2805. Prepublished online as a Blood First Edition Paper on December 19, 2006; DOI 10.1182/blood-2006-10-049312.
GENE THERAPY In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance1 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 2 Immunopathogenesis of Liver Infections Unit, San Raffaele Scientific Institute, Milan, Italy; 3 Vita Salute San Raffaele University, Milan, Italy; 4 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA
Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFN  ß response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNß, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type–specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.
Lentiviral vectors (LVs) are a promising candidate system for therapeutic gene transfer. Because of their capacity to transduce nondividing cells and stably integrate a gene expression cassette of relatively large size and complexity, LVs have significant potential for achieving long-term expression of a therapeutic molecule. Several groups, including our own, have carried out studies using LV for in vivo gene delivery in rodents.1–5 Efficient gene transfer to the liver could be achieved; however, hepatocytes, which were the main target of the therapy, were transduced at a relatively low efficiency compared with nonparenchymal cells. At a low vector dose, this effect was particularly pronounced. While a high frequency of Kupffer cells (KCs) were found to be vector positive, only a small fraction of hepatocytes were transduced. Interestingly, by increasing the concentration of injected vector a threshold is reached in which hepatocyte transduction becomes dose responsive, and improved hepatocyte gene transfer is achieved. This may be due to the requirement for saturating the particle-clearance systems of the sinusoid-lining cells in blood-filtering organs.6 Nonetheless, a better understanding of the rate-limiting factors in transduction would help to improve both the dose-effect relationship and risk-benefit ratio of systemic LV administration. A high incidence of transgene-specific immunity has also been observed in studies using LVs for in vivo gene delivery.1–5 This response resulted in elimination of transduced cells and/or neutralization of the transgene product, and ultimately negated the benefits of gene transfer. The role of transgene expression within antigen-presenting cells (APCs) of the hematopoietic system has been identified as a factor contributing to the development of antitransgene immunity.1 However, the effect of innate immune responses on LV-mediated gene transfer has not been examined in detail.
Recent studies have investigated the consequences of exposing human dendritic cells (DCs) in culture to wild-type HIV, the parent virus of many LVs.7,8 These works indicate that HIV activates a subset of DCs, plasmacytoid DCs (pDCs), through engagement of toll-like receptor 7 (TLR7), a pattern-recognition receptor (PRR) for single-stranded RNA (ssRNA). In response to HIV, pDCs produce high levels of IFN The studies of HIV, although informative, were carried out in vitro, and do not necessarily reflect the in vivo conditions of a systemically administered LV. In addition, LVs are nonreplicating, hybrid vectors. They lack all viral accessory proteins associated with virulence and pathogenesis, and have been pseudotyped by the envelope of an unrelated virus. This affects their biodistribution, target cell binding, and entry. Thus, the aforementioned studies do not necessarily provide insight into the potential consequences of LV-mediated activation of the innate immune system.
To address these issues, we have undertaken studies to monitor the innate host response following in vivo LV delivery to mice. Our results provide new findings indicating that LVs activate an IFN
Vector production Third-generation LVs were produced by Ca3PO4 transfection into 293T cells. Supernatants were collected, passed through a 0.22-µm filter, and purified by ultracentrifugation as described.10 Titer was estimated on 293T cells, and vector particles were measured by HIV-1 Gag p24 antigen immunocapture (NEN Life Science Products, Boston, MA). Vector infectivity was calculated as the ratio between titer and particle. For concentrated vesicular stomatitis virus (VSV) pseudotyped vector, titer ranged from 0.7 to 1.5 x 1010 transducing units (TU)293T/mL, and particles ranged from 90 to 150 µg/mL p24. For concentrated gp64 pseudotyped vector, the titer was 109 TU/mL, and the particles were 100 µg/mL p24. In vivo vector administration
Balb/c, C57BL/6, 129sv, and Nu/Nu mice were purchased from Charles River Laboratories (Milan, Italy), and IFN Quantitative PCR and RT-PCR DNA from cells and tissues was extracted by using the Blood & Cell Culture DNA Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA from cells was extracted by using Tri Reagent (Sigma, St Louis, MO), according to the manufacturer's instructions. Vector copies per genome (C/G) were quantified by quantitative polymerase chain reaction (Q-PCR) using the primer and probe set previously described.1 Copies per genome (C/G) were calculated by the following equation: (ng LV/ng endogenous DNA) x (no. of LV integrations in the standard curve). Reverse transcription (RT) was carried out on 2 µg total RNA from the livers and spleens of treated mice using the random hexamers protocol of the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions using the primer/probe set previously described.1 All reactions were carried out in triplicate in an ABI Prism 7900 (Applied Biosystems, Weiterstadt, Germany). Cytokine expression analysis
The RNase protection assay for quantitation of mRNA was performed as previously described.11 The mouse interleukin-1 Immunohistochemistry For immunofluorescence, tissues were prepared as previously described13 and stained with the indicated antibodies. Images were visualized with a Zeiss Axioskop2 microscope using triple laser confocal microscopy with a Zeiss Plan-Neofluar 20x/0.5 numerical aperture objective lens and a Zeiss W-PI 10 x 0.23 objective lens as eyepiece (Zeiss, Arese, Italy). Images were acquired using a Radiance 2100 camera (Bio-Rad, Segrate, Italy) and LaserSharp 2000 acquisition software (Bio-Rad). Morphometric analysis was used to determine the frequency of green fluorescent protein (GFP)–positive hepatocytes. Confocal images were imported into ImageJ software (http://rsb.info.nih.gov/ij/). Three random liver fields were measured per mouse. We scored the total number of TOPRO-3–positive large nuclei, which were taken to be hepatocytes, to determine the total number of hepatocytes per field. Since most hepatocytes are binucleated, caution was taken to prevent double scoring of single hepatocytes. GFP-positive hepatocytes were scored based on 488:515 excitation-emission signal, and morphology to distinguish them from nonparenchymal cells. Frequencies were calculated based on the counting of a minimum of 1000 hepatocytes per mouse. Plasmacytoid DCs and CD11b+ splenocyte isolation Spleens were excised from Balb/c mice and incubated with collagenase D to obtain a unicellular solution. CD11b splenocytes and pDCs were isolated by commercially available kit according to the manufacturer's instructions (no. 130-049-601 and no. 130-091-263, respectively; Miltenyi Biotec, Bergisch Gladbach, Germany). Per experimental analysis, 5 to 6 spleens were used.
IFN-
GFP-specific IFN-
LV entry and RT occurs rapidly after intravenous administration, but vector DNA is quickly lost Balb/c mice were intravenously injected via tail vein with 10 µg of HIV-1 Gag p24 equivalents of VSV glycoprotein pseudotyped third-generation LV (VSV.LV)–encoding enhanced GFP under the control of the ubiquitously expressed phosphoglycerate kinase (PGK) promoter. This vector dose was chosen based on previous experience, which suggested that at this concentration the threshold for achieving significant hepatocyte transduction starts to be overcome. Q-PCR analysis was used to follow transduction kinetics. At 4 hours after injection high levels of LV genomic RNA and proviral DNA could be detected within the liver and spleen (Figure 1A-B). In contrast, no vector DNA or RNA could be detected at the same and later time points in animals injected with a control LV, which was assembled without a viral envelope (bald.LV). The inability to detect vector sequences from mice treated with bald.LV suggests that in VSV.LV-treated animals the RNA and DNA content was not from extracellular vector particles or plasmid DNA trapped within the organs, but indeed due to vector particles that had undergone cell entry and RT. An additional Q-PCR analysis, using primers and a probe specific for plasmid backbone sequences, did not detect significant levels of transfer plasmid (data not shown), and thus further confirmed that the detected vector DNA within the liver and spleen was the result of LV transduction.
By 24 hours, RNA content had sharply declined, but by 72 hours, RNA levels were again elevated, indicative of vector-mediated transcription. Interestingly, these transduction kinetics were consistent with those reported for HIV infection in vitro,15 and indicate that cellular entry and RT of the vector occurs rapidly following in vivo administration. Vector DNA content, although initially high, began to diminish soon after RT. By 72 hours after injection, vector content had declined 3-fold from peak detection levels within the liver (Figure 1C). The mechanism responsible for this initial loss of vector DNA was unclear, however; following this early and rapid decline, there was a delayed, but near complete clearance of LV DNA. This second phase was expected, as it is well established that intravenous delivery of a ubiquitously expressed LV results in immune-mediated destruction of transduced cells, and ultimately, the loss of vector DNA.
In vivo administration of LV activates an IFN To measure the innate response to LV, we used a RNase protection assay (RPA) to monitor changes in the hepatic and splenic expression of cytokines and cytokine-responsive genes known to be produced by activated cells of the innate immune system.16 RPA provides a sensitive method for detecting changes in the transcriptional profile of multiple genes within a tissue. The liver and spleen are the most well transduced tissues following intravenous injection of LVs, and are major targets of systemic gene therapy as well as important sites of innate host protection.
Balb/c and C57BL/6 mice were treated with 10 µg of LV. Within 4 hours of vector administration there was a more then 8-fold induction of OAS, a downstream product of IFN
Induction of the IFN ß response is dependent on LV infection
To improve vector titer and broaden cell tropism, the VSV envelope has routinely been used for pseudotyping LVs.17,18 The high transduction efficiency of this vector, in particular for cells of the innate immune system such as DCs and macrophages, may be responsible for the robust induction of IFN
To determine whether VSV.G was responsible for the observed IFN Since the transduction efficiency of a vector, particularly for hematopoietic-lineage cells, may influence the innate stimulatory capacity of that vector, we initially set out to evaluate the transduction profile of gp64.LV. Balb/c mice were administered 15 µg of gp64.LV by IV, and killed at 4 hours and 7 days after injection. A higher vector dose was used for these experiments because of the lower infectivity (TU/p24 ratio) of gp64.LV compared with VSV.LV. Indeed, even at the higher dose, transduction levels were still lower with gp64.LV than those obtained with VSV.LV. Nonetheless, gp64.LV was able to mediate significant levels of gene transfer (Figure 3A-B) in both the liver (0.7 ± 0.07 C/G) and spleen (0.9 ± 0.2 C/G). Confocal fluorescence microscopy analysis found that GFP expression was confined predominantly to hepatocytes in the liver and stromal cells in the spleen (Figure 3B). However, a fraction of GFP-positive cells in both the liver and spleen were found to costain for the hematopoietic-specific marker CD45, and thus indicates that gp64.LV also transduces hematopoietic cells, albeit to a lesser extent than VSV.LV.
To measure the innate immune response to gp64.LV, RPA was carried out (Figure 3C). Within 4 hours of injection, there was a 4-fold induction of OAS in both the liver and spleen, and a 14-fold induction of TNF- expression in the liver. Thus, the IFNß response to LV is not specific to the VSV envelope, but likely is a function of other vector components.
To rule out the possibility that contaminants, such as plasmid DNA or endotoxins, which can be carried over from vector production, were directly responsible for the activation of the IFN
A second control vector, bald.LV, which was not pseudotyped with a viral envelope, was administered to Balb/c and C57BL/6 mice. As noted, vector RNA and proviral DNA could not be detected in mice treated with 10 µg of bald.LV, and shows that the early stages of vector transduction does not occur with this vector. Here again, we did not observe induction of IFN
Overall, these results demonstrate that activation of the IFN
pDCs, not macrophages or myeloid DCs, produce high levels of IFN
Having established that LV transduction triggers IFN
To determine which cells of the innate immune system were responding to LV, we monitored expression of IFN
Cell were exposed to either PBS or 600 ng p24/mL of VSV.LV or bald.LV, a concentration that corresponds to approximately 10% of the vector dose used in vivo. Higher vector doses were not used because of the possible confounding effect of VSV-mediated toxicity to the cells. Supernatants were collected at 48 hours after exposure. As shown in Figure 4C, pDCs produced high levels of IFN and TNF- in response to VSV.LV, but not to bald.LV. In contrast, LV did not trigger cytokine production from CD11b+ splenocytes. Interestingly, FACS analysis found that less than 1% of pDCs were GFP positive (Figure 4B), indicating that transduction is not required for triggering pDC production of IFN. This may explain how gp64.LV, which has a low infectivity for hematopoietic cells, could trigger an IFNß response. Overall, our results are consistent with those recently reported for wildtype HIV,7,22 and suggest that the IFNß response seen in vivo following LV administration may be primarily mediated by pDCs. The type I IFN response limits the efficiency and stability of LV-mediated gene transfer
IFN
To address these questions, we treated IFN
LV content was measured in the liver and spleen of treated animals by Q-PCR (Figures 5B,S2B). At 24 hours after injection, IFN ßR–/– mice had almost 3-fold higher vector content within the liver compared with normal mice (19 ± 5 and 7 ± 0.8 C/G, respectively). This marked difference, early after vector administration, suggests that the IFNß response negatively impacts the initial steps of LV transduction.
To further investigate this phenomenon, the GFP expression profile of the liver and spleen was analyzed (Figures 5C,S2A). As seen in Figure 5C, there was a striking difference in the pattern of GFP expression between the 2 treatment groups within the liver. In normal mice, at the dose tested, vector expression occurred predominately within KCs. Only a small fraction of hepatocytes were GFP positive, while in IFN
In normal 129sv mice, by day 36, vector content in the liver dropped below 0.3 C/G (Figure 5B). GFP-positive cells were found in some mice, however, at low frequency, and predominately within nonparenchymal cells. In contrast, the LV content within the liver of IFN
To better understand the immunologic status of GFP in the 2 treatment groups, ELISPOT analysis for IFN-
In normal mice, transgene expression is progressively down-regulated and ultimately cleared by the adaptive immune response. Thus, it is difficult to distinguish any direct effect of the IFN
Here, we report that intravenous administration of late-generation LVs in mice induces a rapid and transient IFN ß response in both the liver and spleen. This supports previous studies carried out with human cells in vitro, which demonstrated that HIV and LV can trigger activation of DCs.7,8,25 Our work extends these findings by providing insight into the effect of this response on LV gene transfer. Unexpectedly, we found that IFNß strongly inhibits transduction efficiency, specifically within the liver, and contributes to immune-mediated clearance of transduced cells.
Induction of the IFN
Several intracellular and endosomal PRRs have been identified, including TLR7 and TLR9, which recognize ssRNA and unmethylated CpG DNA, respectively.27–32 Both HIV and LV carry genetic information as ssRNA and enter the cell through an endosome-mediated pathway. Thus, both have the potential to trigger intracellular PRRs. Since TLR7 is restricted to endosomes, and primarily expressed in pDCs, this would explain our findings that an infectious LV particle is required for innate activation, and that pDCs but not other hematopoietic cells respond to LV. Indeed, Bhardwaj and colleagues have carried out detailed studies demonstrating that HIV activates pDCs through TLR7 signaling.7 However, it has been shown that endogenous RT occurs within the viral particle itself;33 thus, we cannot rule out the possibility that TLR9 and/or an alternate DNA PRR31,32 is also triggered by LV. Indeed, preliminary work from our lab indicates that a TLR7/9 antagonist is not sufficient to prevent LV from inducing an IFN
Our results indicate that the IFN
Only a few studies of wild-type HIV have examined the effects of IFN
Vector content and transgene expression persisted for more than 5 weeks after injection in all LV-treated IFN
Although we found that IFN
From the latter perspective, our data suggests that the transient IFN
An alternative or complementary explanation is that the improved transduction efficiency of LV, due to the absence of IFN
The work presented here provides the first detailed in vivo analysis of the innate host response to LV. We found that LV induces a strong IFN
Contribution: B.D.B. designed and performed research, analyzed data, and wrote the paper; G.S. designed and performed research, analyzed data, and edited the paper; A.A., E.H., L.S.S., and A.Z. performed research and data analysis; M.G.R. and L.G.G. designed research, analyzed data, and edited the paper; and L.N. designed research, analyzed data, and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. B.D.B. and G.S. contributed equally to this work. Correspondence: Luigi Naldini, San Raffaele Telethon Institute for Gene Therapy, Via Olgettina 58, 20132 Milan, Italy; e-mail: naldini.luigi{at}hsr.it.
The authors would like to thank Matteo Iannacone for advice and assistance. This work was supported by grants from Telethon and the Italian Ministry of Scientific Research (L.N.). B.D.B. is the recipient of a Natural Science and Engineering Research Council of Canada fellowship.
Submitted October 7, 2006; accepted November 22, 2006.
Prepublished online as Blood First Edition Paper, December 19, 2006
DOI: 10.1182/blood-2006-10-049312
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Top
Abstract
Introduction
, mTNF-
) and spleen (
) of VSV.LV-treated Balb/c mice. Quantitative RT-PCR (Q-RT-PCR) was used to measure GFP RNA. All samples were normalized to murine GAPDH. Values are presented as the means ± SEM for 3 mice per group. (B) Quantification of LV DNA from the liver (
) or 10 µg of VSV.LV (
).
indicates PBS; 
