Effective repeat administration of adenovirus vectors following intranasal or intravenous delivery is hindered by a strong neutralizing antibody response to the vector. Intramuscular administration of adenovirus vectors elicited a neutralizing antibody response that peaked between 14 and 21 days after infection. However, effective repeat intramuscular administration of adenovirus vectors was not hindered by the presence of neutralizing antibodies in the serum. Surprisingly, β-galactosidase expression in the skeletal muscle of immunized mice was equivalent to that observed in control mice. As expected, these serum neutralizing antibodies effectively blocked repeat administration of adenovirus vectors when delivered via the intravenous route. These results were observed in both C57BL/6 and Balb/c mice and thus do not appear to be strain specific. Successful repeat administration of adenovirus vectors to skeletal muscle has significant implications for the use of adenovirus vectors clinically and for increasing the safety and efficacy of adenovirus vector gene delivery.
A major advantage of adenovirus vectors for gene therapy is that they efficiently infect many differentiated cell types without requiring cell division. Moreover, this tissue tropism has been further expanded by modifications to the virus capsid components.1234 In addition, adenovirus vectors have a relatively large carrying capacity (ie ample space for one or more therapeutic transgenes) and they can be grown to high titers and purified with relative ease.
A major limitation of the adenovirus vector system for treatment of chronic genetic disease is that the vectors are highly immunogenic. Transient expression of immunogenic transgenes is often observed in animal models.56 However, long-term expression of non-immunogenic transgenes has been observed, suggesting that the cellular immune response to low-level viral gene products is not sufficient to destroy vector-transduced cells.78910 An additional immunological limitation is the development of adenovirus-specific neutralizing antibodies following primary administration, which hinder repeat administration of the vector.111213 Delivery of adenovirus vectors to immune competent mice by intravenous (i.v.),7 interperitoneal, intratrachael,1213 or via direct injection into the pancreas14 resulted in the production of neutralizing antibodies and a block to repeat administration.
For adenovirus vectors to be effective in treating chronic genetic diseases, the cell-mediated and humoral immune responses elicited by the vector may need to be circumvented. The development of the neutralizing antibody response to adenovirus appears to require the presentation of exogenous viral antigens by major histocompatibility complex (MHC) class II molecules, as MHC class II knockout mice fail to develop neutralizing antibodies. MHC class II molecules are necessary for the activation of CD4+ T cells, which function in the formation of neutralizing antibodies that hinder repeat administration of Ad vectors.11 For this reason, several investigators have targeted T cell activation in attempts to reduce the humoral response against adenovirus vectors.
Experiments using immunosuppressant drugs or antibodies specific for CD4,131516 CD40 ligand,1417 or CTLA4Ig18 have demonstrated reductions in the adenovirus-specific humoral immune response and prolonged transgene expression. Although these results have been encouraging, there is a risk associated with systemic immune suppression in the clinical setting.
Neutralizing antibodies directed against one adenovirus serotype do not block infection by another adenovirus serotype.19202122 Thus, serotype switching has enabled successful repeat dosing.232425 A disadvantage of this strategy is that it requires multiple vectors, adding considerable cost and complexity to gene therapy.
Modification of the adenovirus capsid or genome is an alternative approach that may enable circumvention of the humoral immune response against the vector. Expression of the adenovirus E3 region from an adenovirus vector in the Gunn rat reduced the anti-adenovirus humoral immune response and enabled successful repeat administration of the vector.26 Another potential target for decreasing the humoral response is lymphotoxin α. Adenovirus vector delivery to mice that lack both tumor necrosis factor α and lymphotoxin α displayed a marked reduction in neutralizing antibody production and efficient repeat dosing was observed.27 Recently, it was demonstrated that a vector carrying a deletion in the E4 region displayed a decreased humoral response following intratracheal administration.28 Specifically, the TH2 response to the E1, E4-deleted adenovirus vectors was reduced relative to the E1-deleted control. This resulted in a decrease in the TH2-dependent, IgG1 adenovirus-specific neutralizing antibody titers.
In this article, we tested the hypothesis that repeat administration of adenovirus vectors to the muscle may be possible without immune suppression or viral modification. We reasoned that the concentration of adenovirus-specific neutralizing antibodies in the muscle may be considerably lower than the concentration in the serum and thus permit effective multiple dosing to the muscle under conditions that do not allow multiple dosing to the liver via the intravenous (i.v.) route.
Kinetics of adenovirus-specific neutralizing antibody response
To determine the kinetics of adenovirus neutralizing antibody production following intramuscular (i.m.) delivery, mice were immunized with 1 × 1010 particles (pu) of three different adenovirus vectors (Table 1). Serum samples were taken at various times after infection and adenovirus neutralizing antibody titers were measured. The neutralizing antibody response was detectable at 10 days and peaked between 14 and 21 days after infection (Figure 1). Adenovirus neutralizing antibody titers dropped off significantly by day 56 (data not shown).
Dose-response analysis of neutralizing antibody production and repeat administration of adenovirus vectors
Development of the humoral response to adenovirus infection is dependent on the dose293031 and route of administration.3233 To determine the minimal dose of vector that results in the production of neutralizing antibodies when delivered by the i.m. route, mice were immunized with escalating doses of an adenovirus vector that expressed luciferase from the RSV promoter. Doses ranged from 102 to 1010 pu (Figure 2a). Neutralizing antibodies were not detectable in mice immunized with <107 virus pu. The first evidence of neutralizing antibody production was at a dose of 107 pu of AdRSV.L (Figure 2b). This was also the minimum dose where detectable luciferase expression from the vector was observed in muscle tissue (Figure 2c). An immunizing dose of 108 pu of AdRSV.L resulted in an increase in both luciferase expression (Figure 2c) and in neutralizing antibody production (Figure 2b). The efficiency of repeat administration was analyzed by measuring β-galactosidase expression in the livers of mice that received i.v. injections of AdZ, 14 days after i.m. immunization. As expected, effective repeat administration of the vector correlated with the adenovirus-specific neutralizing antibody concentration in the serum. After immunization with <107 pu, effective repeat administration was observed. However, an immunizing dose of 108 reduced the efficiency of repeat dosing. Increasing the immunizing dose of vector resulted in increases in the neutralizing antibody titers and reductions in the efficiency of repeat administration. For example, after an immunizing dose of 1010 pu of AdRSV.L, β-galactosidase expression was not detectable in the liver following repeat administration (Figure 2d).
Effective repeat administration to mouse skeletal muscle
To determine if successful repeat administration of adenovirus vectors to the muscle was possible, C57BL/6 mice were immunized with i.m. injections of 1010 pu of either AdRSV.L or AdNull. Two weeks later, repeat administration via the i.v. and i.m. routes was performed with AdZ. As expected, anti-adenovirus neutralizing antibody responses were observed (Figure 3b) and repeat i.v. administration was unsuccessful (Figure 3c). β-Galactosidase activity was not detected in the livers of immunized mice after i.v. administration of AdZ. In contrast, when AdZ was administered by the i.m. route 2 weeks after immunization, efficient delivery was achieved in the muscle. There was no difference in β-galactosidase activity in the immunized mice compared with the naive controls (Figure 3d). This result was unexpected, as previous reports have shown that repeat administration by i.v.,7 intratrachael1213 or direct injection into the pancreas14 did not result in efficient infection.
C57BL/6 mice do not mount strong immune responses to some foreign transgenes. Persistent transgene expression has been observed when human α1-antitrypsin7 or human factor IX10 were expressed from adenovirus vectors in C57BL/6 mice. In contrast, Balb/c mice mounted strong immune responses to both α1-antitrypsin and factor IX and transgene expression was transient.710 To determine if effective multiple dosing to the muscle was strain specific, we analyzed repeat delivery in Balb/c mice. Our results in Balb/c mice mirrored those observed in C57BL/6 mice (Figure 4). Secondary i.m. administration 14 days following primary immunization resulted in efficient transduction of the muscle, with β-galactosidase expression equivalent to that observed in non-immunized controls (Figure 4c). Neutralizing antibodies that were present in the serum (Figure 4a) blocked repeat administration to the liver when virus was administered via the i.v. route (Figure 4b). These results indicate that the ability to repeat dose in the mouse skeletal muscle with adenovirus vectors is not strain dependent.
Increased skeletal muscle specificity by pre-immunization
The differential ability of serum neutralizing antibodies to inhibit adenovirus transduction following i.v. versus i.m. delivery suggests a role for immunization as a method to increase skeletal muscle specificity. Immunization could reduce potential toxicity associated with vector spread from the skeletal muscle through the circulation to non-target tissues. To analyze this in a preclinical setting, mice were injected with 1 × 1010 pu of AdhβActin.L or with 1 × 1011 pu of AdZ in the gastrocnemius muscle. Twenty-four hours later, luciferase or β-galactosidase activities were measured in the injected muscle and in the liver. As expected, most of the enzymatic activity was observed in the injected muscle (Figure 5a and b). However, a significant level of enzymatic activity was observed in the liver of these mice (Figure 5c and d). This indicates that vector leaking from the injected muscle enters the circulation and transduces the liver and presumably other tissues. When animals were immunized with 1 × 1010 pu of AdNull 14 days before administration of AdhβActin.L or AdZ, there was no reduction of enzymatic activity in the injected muscle. Importantly, no increase in enzymatic activity was observed in the liver of these immunized animals (Figure 5c and d). This result is consistent with the theory that the virus was neutralized by circulating antibodies upon entering the circulatory system, thus increasing the muscle selectivity of the administration.
The hypothesis that repeat administration of adenovirus vectors to the skeletal muscle may be possible without immune suppression was tested. We reasoned that the concentration of adenovirus-specific neutralizing antibodies in the muscle may be considerably lower than in the serum, thus permitting effective multiple dosing to the muscle. Although further investigation will be required to establish the mechanism for the observed effect, there is a clear differential between the ability to redose by direct injection into the muscle versus administration via the i.v. route. Immunization with 1 × 1010 pu of adenovirus vector permitted effective repeat administration by direct injection into the skeletal muscle but not to the liver following i.v. delivery.
There have been several reports of effective repeat administration of adenovirus vectors in immunocompetent animals in the absence of any immune-modulating therapies. In one study, subretinal injection of an adenovirus vector resulted in minimal anti-adenovirus antibody production relative to a subcutaneous injection and successful repeat administration was observed.34 However, effective repeat dosing in this organ is probably a reflection of the immune privileged status of the anterior chamber of the eye.35 Successful repeat administration of adenovirus vectors has also been observed in gene therapy models for cancer.3637 Four i.m. administrations of an adenovirus vector given over a 1 week period were shown to induce a strong neutralizing antibody response, that did not completely abrogate expression from a second adenovirus vector administration.37 However, transgene expression was not quantified in this study to determine the relative efficiency of repeated intratumoral delivery under these conditions. In another report, neutralizing antibodies were induced by intranasal immunization of mice with wild-type adenovirus type 5 one month before intratumoral administration. Immunization reduced the peak levels of transgene expression in the tumor by only 2.4-fold, suggesting that neutralizing antibodies did not efficiently inhibit repeat administration to the tumor.36 These studies suggest that direct injection of adenovirus vectors into a solid tissue mass can overcome the neutralizing effect of antibodies directed at adenovirus capsid components. Perhaps the structural integrity of the tumor or the extracellular matrix surrounding the tumor presents a barrier to the neutralizing antibodies. Alternatively, immunization with wild-type adenovirus, which results in the expression of E3 products, may have inhibited components of the humoral immune response.26 A third possibility is that the tumor cells themselves actively inhibited the effectiveness of the humoral response, permitting successful repeat dosing in these systems.
Repeat administration of adenovirus vectors has also been investigated following localized delivery to the artery wall. Efficient delivery to the left ventricle by the percutaneous transluminal method was demonstrated in a canine model.38 As an adenovirus vector expressing LacZ was used in these experiments, transgene expression was transient. Repeat administration at 14 days after infection led to an increase in β-galactosidase expression, although this level was significantly reduced relative to the initial injection. This reduced expression upon repeat administration may have been due to neutralizing antibodies that were detected in the serum, a cellular immune response directed at either viral gene products or β-galactosidase, or a combination of these factors. Additional studies will be required to differentiate among these possibilities. In a similar fashion, the cellular immune response against β-galactosidase may have limited repeat administration following delivery to the canine femoral artery39 and to the rat carotid artery.40 However, considering our findings it is conceivable that effective repeat administration to the myocardium and to the arterial wall will be possible. This will be an important area for future research.
Recently, there has been tremendous excitement regarding the use of adenovirus vectors expressing angiogenic growth factors for the treatment of vascular diseases. Intramuscular administration of adenovirus vectors expressing vascular endothelial growth factor (VEGF) stimulated angiogenesis in the setting of hindlimb ischemia in rats41 and nonobese diabetic (NOD) mice.42 These results suggest that expression of angiogenic genes using adenovirus vectors may be an effective therapy for patients with peripheral vascular disease. Therapeutic angiogenesis also offers great promise for coronary artery disease. Both fibroblast growth factor-5 and VEGF were shown to induce collateral vessel development in the ischemic porcine myocardium following adenovirus-mediated gene transfer.4344 These treatments resulted in significant improvement in myocardial perfusion and function. The ability to repeat dose in the clinic for both peripheral and coronary vascular disease may improve the efficacy of gene therapies. In addition, effective treatment of recurrent ischemic disease will require repeat delivery.
It appears that adenovirus vectors will have utility for diseases such as cancer and vascular disease that require transient transgene expression. We have demonstrated that intramuscular delivery of an adenovirus vector to immunized animals resulted in high level transgene expression at day 1 after secondary administration. The persistence of the readministered vector is not clear and will likely be a function of the cellular immune response to the vector and transgene product, promoter shutoff and other factors. Further experiments will be required to evaluate the persistence of non-immunogenic transgene expression in this model. However, even transient high-level gene expression may yield a therapeutic effect. For example, Fas ligand expressed from an adenovirus vector effectively kills cells in vitro within 24 h454647 and in vivo within 3 days.48 This has clear implications for cancer gene therapy.
Duchenne muscular dystrophy, hemophilia A and hemophilia B are examples of genetic diseases where long-term transgene expression and repeat administration will probably be necessary to achieve success in the clinic. The recent evidence for long-term expression of non-immunogenic transgenes with adenovirus vectors78910 taken together with our demonstration of effective repeat delivery to the muscle suggest that adenovirus vectors may be useful in the treatment of genetic diseases in the future.
Up to 50% of humans have serum neutralizing antibodies to adenovirus type 5.4049 This presents a potential problem in that neutralizing antibodies may limit the effectiveness of adenovirus vectors for gene therapy in these individuals. However, our results suggest that i.m. administration of adenovirus vectors may be effective even in previously exposed patients. Of course, clinical studies will be necessary to determine whether these findings in the mouse are predictive for humans.
We have demonstrated that adenovirus vector leaking from an injected skeletal muscle can spread to distal organs, including the liver. This example illustrates the idea that the leakage of a therapeutic vector from the skeletal muscle could result in deleterious side-effects as a result of gene expression in non-target tissues. Interestingly, pre-immunization prevented liver expression while preserving target tissue expression at levels equal to that of non-immunized animals. Similar findings have been reported by Bramson et al.36 They observed a dramatic decrease in virus dissemination to the liver in immunized mice following intratumoral administration with only a minor effect on delivery to the tumor. These results suggest that immunization may be an effective strategy for targeted delivery. This may be especially relevant for previously exposed patients that have circulating anti-adenovirus neutralizing antibodies. A safety advantage would be realized if misinjected vector or leaky vector entering the circulation were neutralized by vector-specific neutralizing antibodies. Thus, our results suggest repeat delivery to the muscle can result in both increased specificity and increased efficacy of gene therapy.
Materials and methods
The E1-, E3-deleted adenovirus vector, AdZ, expresses β-galactosidase from the cytomegalovirus (CMV) promoter pointing left and carries the simian virus 40 poly A sequences from an expression cassette inserted at the site of the E1 deletion. AdF and AdL are similar to AdZ except that the CMV promoter drives the expression of green fluorescent protein and luciferase, respectively. AdNull contains the CMV promoter and simian virus 40 poly A sequences in place of the E1 region and does not express any transgenes. AdhβActin.L expresses luciferase from an expression cassette containing the 4.3 kb human β-actin promoter pointing left and the simian virus 40 poly A sequences. pAdRSV.L and AdMCK.L drive luciferase expression from the Rous sarcoma virus or the 3.3 kb muscle creatine kinase promoter, respectively. All adenovirus vectors contain the dl324 E3 deletion and were generated using shuttle vectors as previously described.650 Briefly, the shuttle vectors were linearized at a unique restriction site adjacent to the left end inverted terminal repeat (ITR) and cotransfected into 293 cells with ClaI-digested AdLacZ DNA. Virus generated by recombination between the shuttle vector and the adenovirus DNA was plaque purified and propagated on 293 cells.51 Viruses were purified from infected cells at 2 days after vector administration by three freeze–thaw cycles followed by three successive bandings on CsCl gradients. Purified virus was dialyzed against a buffer containing 10 mM Tris, pH 7.8, 150 mM NaCl, 10 mM MgCl2 and 3% sucrose and stored at −70°C until use. All viruses were tested and found to have replication-competent adenovirus (RCA) levels of less than 1 in 1 × 107 plaque forming units (p.f.u.).
Female Balb/c and C57BL/6 mice were obtained from Charles River (Wilmington, MA, USA) at 6 to 8 weeks of age. Before administration mice were anesthetized with a 0.1 ml intraperitoneal injection of ketamycin and rhompin (three parts water one part ketamycin/rhompin dilution). Vectors were administered intramuscularly in a 50 μl volume. At the indicated times after administration, mice were given an intraperitoneal injection of a terminal dose of anesthetic. The gastrocnemius muscles and livers were removed and washed quickly with PBS and flash frozen in liquid nitrogen, ground with a mortar and pestle, aliquoted, and stored at −80°C until use. Intravenous administration was performed by exposing the right jugular vein after making a supraclavicular incision and the vectors were injected by using a 30-gauge needle over a period of 2 min.
Neutralizing antibody assay
Neutralizing antibody titers were determined by analyzing the ability of serum antibody to inhibit infection of AdF on AE25 cells. AE25 cells were inoculated at 2 × 104 per well on flat bottom 96-well plates and grown for 18 to 24 h at 37°C. A series of two-fold dilutions of the serum samples was incubated with AdF at a multiplicity of infection of 3 focal-forming units per cell for 1 h at 37°C in minimal DMEM medium. This mixture was incubated with AE25 cells for 1 h at 37°C, 100 μl of complete medium was added and the cells were cultured overnight. The neutralizing antibody titer was scored as the reciprocal of the last dilution where a 50% reduction in green cells (infected cells) was observed.
Pulverized muscle or liver tissue was lysed in 1 × Reporter Lysis Buffer (Promega, Madison, WI, USA) and protein determinations were made using the Bradford reagent. Protein samples were used to measure β-galactosidase activity with the β-galactosidase reporter gene assay system (Tropix, Bedford, MA, USA).
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We thank Jennifer Lee, Peter Genis and Emanuel Haney Jr for support with animal surgery, vector administration and animal care, Alena Lizonova, Angela Appiah and Lu Qin for their support with virus production and cell culture and Lisa DeBruyne, He Wang, Joan Keiser, David Gordon, Rob Panek, Mike Flynn, Doug Brough, Tom Wickham and Paul Fischer for insightful discussions. We also thank Rena Cohen and Kelly Raygor for preparation of the manuscript.
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Chen, P., Kovesdi, I. & Bruder, J. Effective repeat administration with adenovirus vectors to the muscle. Gene Ther 7, 587–595 (2000). https://doi.org/10.1038/sj.gt.3301137
- gene therapy
- humoral immunity
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