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. 2012 Jun 26;20(9):1713–1723. doi: 10.1038/mt.2012.114

Gene Transfer to the CNS Is Efficacious in Immune-primed Mice Harboring Physiologically Relevant Titers of Anti-AAV Antibodies

Christopher M Treleaven 1, Thomas J Tamsett 1, Jie Bu 1, Jonathan A Fidler 1, S Pablo Sardi 1, Gregory D Hurlbut 1, Lisa A Woodworth 1, Seng H Cheng 1, Marco A Passini 1, Lamya S Shihabuddin 1, James C Dodge 1,*
PMCID: PMC3437584  PMID: 22735381

Abstract

Central nervous system (CNS)-directed gene therapy with recombinant adeno-associated virus (AAV) vectors has been used effectively to slow disease course in mouse models of several neurodegenerative diseases. However, these vectors were typically tested in mice without prior exposure to the virus, an immunological scenario unlikely to be duplicated in human patients. Here, we examined the impact of pre-existing immunity on AAV-mediated gene delivery to the CNS of normal and diseased mice. Antibody levels in brain tissue were determined to be 0.6% of the levels found in systemic circulation. As expected, transgene expression in brains of mice with relatively high serum antibody titers was reduced by 59–95%. However, transduction activity was unaffected in mice that harbored more clinically relevant antibody levels. Moreover, we also showed that markers of neuroinflammation (GFAP, Iba1, and CD3) and histopathology (hematoxylin and eosin (H&E)) were not enhanced in immune-primed mice (regardless of pre-existing antibody levels). Importantly, we also demonstrated in a mouse model of Niemann Pick Type A (NPA) disease that pre-existing immunity did not preclude either gene transfer to the CNS or alleviation of disease-associated neuropathology. These findings support the continued development of AAV-based therapies for the treatment of neurological disorders.

Introduction

Central nervous system (CNS)-directed gene therapy with recombinant adeno-associated virus (AAV) vectors has shown promise as a therapeutic paradigm in several rodent models of neurodegeneration.1,2,3,4,5,6,7,8 However, animals used in these studies were typically immunologically naive to AAV before treatment. In contrast, clinical testing of an experimental AAV-based therapy will likely involve subjects who have had prior exposure to the virus. A significant percentage (e.g., 80% for AAV2/2) of the general population reportedly maintains antibodies to AAV, presumably initiated by pulmonary infection.9,10 Although it has been documented that prior exposure to AAV precludes efficient gene transfer to the visceral organs,11,12 it remains unclear whether pre-existing immunity exerts a similar effect in the relatively “immunoprivileged” CNS. For example, it has been suggested that circulating antibodies may not cross the blood–brain barrier in sufficient quantities to block the infection of CNS target cells.13 Hence, it is of interest to investigate the efficiency of AAV-mediated gene transfer to the CNS of immune-primed rodent models since a number of clinical trials employing AAV-based therapies are being considered to treat neurological diseases.14,15,16,17,18

Previous work conducted in rats has shown that relatively high titers of circulating neutralizing antibodies to AAV capsids can negate AAV2/2-mediated gene expression within the CNS.19,20 Interestingly, preimmunization (even at very high titers) does not appear to impair gene transfer to the CNS for all AAV serotypes (e.g., AAV2/5).19 These findings suggest that highly elevated neutralizing antibody titers against certain viral serotypes might be considered as exclusion criteria for clinical studies involving AAV-mediated gene therapy to brain. The presence of neutralizing antibody titers; however, may not be the most sensitive indicator of prior viral exposure or the best predictor of any subsequent immune response to viral re-exposure.21 For example, a recent survey of serum samples from 70 healthy individuals showed that total anti-AAV8 antibody titers could be measured in all 70 samples, whereas only 33 had a detectable neutralizing titer of ≥1:25. Although neutralizing antibody titers found in humans have been reported for various AAV serotypes,22,23,24 the values for total anti-AAV antibody titers have been less well documented. Additional work is desirable to document total anti-AAV titers against the various AAV serotypes in the general population and determine what levels might potentially impair AAV-mediated gene transfer to the CNS.

Another factor to contemplate when considering the subsequent immune response to delivery of recombinant AAV vectors to the CNS is the anatomical site of injection. For example, the humoral and cellular immune responses after intracerebroventricular (ICV) injection of adenovirus (Ad) vectors is reportedly greater than following delivery into brain parenchyma.25 Understanding the corresponding immune responses generated by recombinant AAV vectors using these different delivery strategies will be informative as a number of emerging experimental therapeutic strategies rely on either intraparenchymal (IP) or cerebrospinal fluid (CSF) (i.e., ICV or intrathecal) vector delivery to treat CNS diseases. From a safety perspective, it is also important to understand whether or not pre-existing immunity to AAV will trigger an enhanced neuroinflammatory response following subsequent vector delivery to the CNS.

Here, we characterized the total anti-AAV2/2 and -AAV2/5 antibody titers in a small sampling of healthy volunteers to determine a physiologically relevant titer to model in mice. Immune-primed mice harboring anti-AAV antibody titers typically found in the general population as well as significantly higher levels were subjected to stereotaxic injections with either recombinant AAV2/2 or 2/5 vectors encoding human insulin-like growth factor-1 (hIGF-1) to investigate the impact of pre-existing immunity on AAV-mediated gene transfer to the CNS. The relative level of AAV-mediated expression of hIGF-1 within the CNS of wild-type mice was evaluated following both IP and ICV vector delivery. Efforts were also made to understand the relationship between serum and brain antibody titer level and to determine whether a pre-existing immunity to AAV exacerbated neuroinflammation following intracranial delivery of recombinant AAV vectors. Finally, the impact of pre-existing immunity on subsequent gene transfer to the CNS in the context of a neurometabolic disease (Niemann Pick Type A (NPA) disease.) was assessed.

Results

Titers of anti-AAV2/5 and -AAV2/2 antibodies in healthy human subjects

Measurements of total anti-AAV2/2 antibody titers in the serum of healthy adult human volunteers showed they were in the range of 1:100–1:51,200 with the majority (70%) displaying a titer of <=1:6,400 (Figure 1). Titers of anti AAV2/5 antibodies were determined to be in the range of 1:100–1:6,400 with the majority (71%) harboring a titer of <1:1,600 (100% of samples were <1:6,400) (Figure 1). We also measured neutralizing anti-AAV antibody titers in these samples to determine whether a linear relationship between the two different types (total versus neutralizing) of antibody titers exists. Similar to what has been reported for serotype 2/821 total anti-AAV antibody titers were detected in 100% of the samples analyzed for serotypes 2/2 and 2/5. In contrast, only 64% (20/31) for serotype 2/2 and 36% (12/33) for serotype 2/5 of the samples analyzed displayed detectable (≥25) neutralizing antibody titers. Surprisingly, no significant correlation was observed between total and neutralizing antibody titers for serotypes 2/2 and 2/5. Nevertheless, these findings confirmed that total anti-AAV antibody titers are a more sensitive indicator of prior AAV exposure and also showed that circulating anti-AAV2/2 and -AAV2/5 antibody titers that are generally below 1:6,400 in a sampling of healthy adults. Assuming that this antibody titer is representative of the larger population, we then sought to determine whether AAV-mediated gene transfer to the CNS is effective in mice with this circulating level of pre-existing antibodies.

Figure 1.

Figure 1

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Anti-AAV antibody titers in human serum. (a) Total and neutralizing anti-AAV antibody titers in human serum for serotype 2/2. (b) Relative distribution of total and neutralizing anti-AAV antibody titers in human serum for serotype 2/2. (c) Total and neutralizing anti-AAV antibody titers in human serum for serotype 2/5. (d) Relative distribution of total and neutralizing anti-AAV antibody titers in human serum for serotype 2/5. Neutralizing anti-AAV antibody titers ≤25 were below the limit of detection. AAV, adeno-associated virus.

Relationship between the levels of anti-AAV antibodies in systemic circulation and the CNS of mice

In contrast to control mice (injected with phosphate-buffered saline (PBS)) that displayed a circulating anti-AAV2/5 antibody titer of 1:200, mice preimmunized with AAV2/5-βgal (following systemic injection of 2.0 E11 drp virus/mouse) exhibited antibody titers of >1:400,000 (Figure 2a). Subsequent stereotaxic delivery of 5 E 9 drps of AAV2/5-hIGF-1 to the CNS of the control (AAV naive) cohort increased the serum antibody titer to >1:400,000 (Figure 2a). Mice that had been primed with AAV2/5-βgal also exhibited a circulating antibody titer of >1:400,000 following stereotaxic injection of AAV2/5-hIGF-1. However, it should be noted that samples were not analyzed beyond this dilution and therefore, the actual titer levels were likely higher.

Figure 2.

Figure 2

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Effect of high circulating total anti-AAV2/5 antibody titers on AAV2/5-mediated transgene expression within the mouse CNS after intraparenchymal (IP) or intracerebroventricular (ICV) vector delivery. Pre-sx = before stereotaxic surgery. Post-sx = after stereotaxic surgery. (a) Serum and (b) brain total anti-AAV antibody levels in naive and immune-primed (intravascular pre-exposure to AAV2/5-βgal) mice versus untreated mice. Human insulin-like growth factor-1 (hIGF-1) levels in the (c) injected and (d) noninjected hemispheres of AAV nave and immune-primed mice after either IP or ICV injection of AAV2/5-hIGF-1 into the brain. Columns not connected by the same letter are significantly (P < 0.01) different from each other. ND = antibody titer (as shown in b) or IGF-1 protein levels (as shown in c and d) were below the limit of detection for the assay. AAV, adeno-associated virus; CNS, central nervous system.

Examination of the brains of mice that were administered AAV2/5-βgal systemically (and exhibiting a circulating titer of >1:400,000) showed the presence of antibody titer levels of 1:800–1:2,430 (Figure 2b) in brain tissue. As expected the antibody levels in the brains of control mice that were treated with PBS were below the level of detection (Figure 2b). Hence, conservatively, the relative antibody levels in the CNS were ~0.6% of those found in the serum. This supports the prevailing notion that only limited amounts of antibodies from systemic circulation cross the blood–brain barrier into the CNS. Subsequent ICV or IP injection of AAV2/5-hIGF-1 into the CNS of control mice (treated systemically with PBS) increased antibody titer levels in the brain to between 1:90–1:810 and 1:810–1:2,430, respectively (Figure 2b). In contrast, brains of mice that had been primed by systemic injections AAV2/5-βgal and then administered AAV2/5-hIGF-1 intracranially displayed antibody titer levels >1:400,000 in brain tissue (Figure 2b). Again, samples were not analyzed for titers beyond this dilution and therefore, the actual titer levels were likely much higher. This finding indicates that presence of very high circulating antibody levels could have a dramatic influence on the subsequent antibody response within the CNS following viral re-exposure.

Efficiency of AAV-mediated gene transfer to the CNS is lower in mice with high pre-existing antibody levels

Examination of hIGF-1 levels in the brains of immune-primed mice following IP or ICV injection of AAV2/5-hIGF-1 showed that the efficiency of gene transfer to the CNS was significantly reduced by the presence of high titers of antibodies. Levels of hIGF-1 in the IP-injected hemisphere were 71% (P < 0.001) lower in immune-primed mice than in nonprimed control animals (Figure 2c). Immune-primed mice administered AAV2/5-hIGF-1 via the ICV route showed a similar large reduction (59%; P < 0.001) in hIGF-1 levels compared to control mice (Figure 2c). As AAV reportedly can undergo axonal transport within the CNS26,27 and CSF flow can occur between the lateral ventricles, hIGF-1 levels in the contralateral hemispheres were also measured. Regardless of the route of injection, a greater reduction in AAV-mediated expression of hIGF-1 was observed in the noninjected hemispheres. The contralateral hemispheres (IP-injected mice) and ventricles (ICV-injected mice) of immune-primed mice displayed a 95% (P < 0.001) and 74% (P < 0.001) reduction in hIGF-1 levels respectively, when compared to control mice (Figure 2d). This observed decrease in transduction activity is consistent with previous reports in rats harboring high neutralizing antibodies to AAV2/2 before vector administration into the CNS.20

AAV-mediated gene transfer to the CNS is effective in mice with low circulating total anti-AAV antibody titers

As a total anti-AAV antibody titer of >1:400,000 in the serum is significantly higher than what might be typically observed in the general population, further studies were performed to ascertain the impact of lower titers (i.e., ~1:6,400) on gene transfer to the CNS. Efforts to elicit specific levels of anti-AAV2/5 antibodies by injecting mice with varying amounts of AAV2/5-βgal were largely unsuccessful (Figure 3). A more manageable approach was to inject mice intravenously with Gamunex (intravenous immune globulins; IVIG). Previous experiments completed in both severe combined immunodeficiency and normal mice have demonstrated that pre-treatment with Gamunex 24 hours before AAV exposure nearly abolished AAV-mediated expression within peripheral organs.28,29 Indeed, in our current study we found that pre-treatment with Gamunex 24 hours before intravenous administration of AAV2/2-hIGF-1 completely abolished transgene expression in the mouse liver (Supplementary Figure S1). Mice injected with Gamunex displayed circulating total anti-AAV antibody titers that were within the range noted earlier in the sampling of human sera. Gamunex-treated mice exhibited anti-AAV2/5 titers of between 1:3,200 and 1:6,400 and anti-AAV2/2 titers of 1:1,600 (Figure 4a). Serum antibody titer levels in control mice (PBS injected) were 1:200. Examination of a subset of the Gamunex-treated mice showed that the corresponding levels of anti-AAV2/2 and AAV2/5 antibodies in the brain were below the limit of detection (Figure 4b). This suggests that mice with serum levels of anti-AAV antibodies titers of ≤1:6,400 are unlikely to harbor detectable amounts of antibodies in the CNS.

Figure 3.

Figure 3

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Total serum anti-AAV2/5 antibody titers detected in mice following intravascular injection of AAV2/5-βgal at various doses. AAV, adeno-associated virus; Ab, antibody.

Figure 4.

Figure 4

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Effect of lower circulating levels of total anti-AAV antibody titers on AAV-mediated gene expression within the mouse CNS after intraparenchymal (IP) or intracerebroventricular (ICV) delivery. Pre-sx = before stereotaxic surgery. Post-sx = after stereotaxic surgery. (a) Serum and (b) brain total anti-AAV antibody levels in naive and immune-primed (by intravascular administration of Gamunex) mice versus untreated mice. Human insulin-like growth factor-1 (hIGF-1) levels in the (c) injected and (d) noninjected hemispheres of naive and immune-primed mice following CNS delivery of AAV2/2-hIGF-1 or AAV2/5-hIGF-1. Columns not connected by the same letter are significantly (P < 0.01) different from each other. ND = antibody titer (as showin in b) or IGF-1 protein levels (as shown in c and d) were below the limit of detection for the assay. AAV, adeno-associated virus; CNS, central nervous system.

Intracranial injection of AAV2/5-hIGF-1 into control mice increased anti-AAV2/2 and -AAV2/5 antibody titers in the CNS (1:90–1:810 after ICV delivery and 1:810–1:2,430 after IP injections) (Figure 4b). Mice administered Gamunex and then injected intracranially with AAV2/5-hIGF-1 displayed similarly low levels of anti-AAV antibodies (1:90–1:2,430) in the CNS (Figure 4b). Consistent with these findings is the observation of comparable levels of expression of hIGF-1 in the brains of Gamunex-treated and control mice (regardless of injection site or route of delivery to the CNS) treated with AAV2/5-hIGF-1 (Figure 4c,d). The presence of systemic anti-AAV antibodies in Gamunex-treated mice also did not negatively affect AAV2/2-hIGF-1–mediated gene transfer to the mouse CNS (Figure 4c,d). Taken together, these results indicate that pre-existing anti-AAV antibody titers that are in the range typically observed in the general population do not negatively impact AAV-mediated gene delivery to the mouse CNS.

Presence of pre-existing immunity to AAV does not trigger an enhanced neuroinflammatory response in the CNS following delivery of recombinant AAV vectors

Astrogliosis, microglial activation, and T-cell infiltration are hallmarks of neuroinflammation30,31 and are often observed during neurodegeneration.32,33,34 Therefore, it was of interest to determine whether a pre-existing immunity to AAV may lead to an enhanced immune response after subsequent delivery of a recombinant AAV vector to the CNS. Mice were evaluated for markers of neuroinflammation at 5 and 30 days post-stereotaxic surgery. Evaluation of GFAP (astrocyte marker), Iba1 (microglial marker), and CD3 (T-cell marker) staining in the CNS of mice with circulating anti-AAV antibody titers of between 1:6,400 and 1:40,000 and that were administered intracranially with AAV2/2-hIGF-1 or AAV2/5-hIGF-1 showed they were not different from mice that had no pre-existing immunity (Figures 5 and 6). Specifically, we observed that the staining pattern and morphology of astrocytes and microglia were comparable to that of AAV naive mice (both at 5 and 30 days after stereotaxic surgery). Examination of T-cell infiltration revealed similar findings with preimmunized and AAV naive mice showing comparable CD3 staining within the CNS after vector delivery (Figures 5 and 6). Histological examination of the brain sections by hematoxylin and eosin (H&E) also showed no difference in histopathology or evidence of cellular infiltration in mice with pre-existing immunity following administration of the recombinant viral vectors (Figures 5 and 6). Hence, a pre-existing immunity to AAV does not trigger an enhanced immune response following AAV-mediated gene transfer to the CNS.

Figure 5.

Figure 5

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Glial cell staining was not enhanced in mice with pre-existing immunity to AAV. GFAP (marker of astrocytes) and IBA1 (marker of microlglia) staining in naive and immune-primed mice following intraparenchymal or intracerebroventricular injection of AAV-hIGF-1. Brain sections were analyzed from mice killed at 5 and 30 days after AAV vector delivery to the CNS. Inset is higher magnification (×20) of area marked by *. Bar = 100 µm, for inset bar = 50 µm. AAV, adeno-associated virus; CNS, central nervous system; GFAP, glial fibrillary acidic protein; hIGF-1, human insulin-like growth factor-1; IBA1, ionized calcium-binding adaptor molecule 1.

Figure 6.

Figure 6

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Neuroinflammation was not enhanced in mice with pre-existing immunity to AAV. CD3 (T-lymphocyte marker) and H&E staining in naive and immune-primed mice after intraparenchymal or intracerebroventricular injection of AAV-hIGF-1. Brain sections were analyzed from mice killed at 5 and 30 days after AAV vector delivery to the CNS. Inset is higher magnification (×20) of area marked by *. Bar = 100 µm, for inset bar = 50 µm. AAV, adeno-associated virus; CNS, central nervous system; H&E, hematoxylin and eosin; hIGF-1, human insulin-like growth factor-1.

AAV-mediated delivery of acid sphingomyelinase into the CNS of ASMKO mice with pre-existing antibodies to the virus is efficacious in correcting the neurometabolic disease

Our experiments completed in wild-type mice demonstrated that clinically relevant anti-AAV antibody titers were not detrimental to subsequent gene transfer to the CNS. To further demonstrate that circulating levels of anti-AAV antibodies are not an impediment to subsequent AAV-mediated gene transfer in the CNS, we carried out efficacy studies in a mouse model of neurometabolic disease following pre-exposure to AAV. Similar to patients with NPA disease, acid sphingomyelinase knockout (ASMKO) mice present with lysosomal accumulation of sphingomyelin, aberrant cholesterol trafficking, and subsequent loss of cellular function in various organ systems including the CNS.35 Previously, we had demonstrated that AAV-mediated gene transfer of human ASM (hASM) to the CNS of ASMKO mice is effective in alleviating the neuropathological accumulation of both sphingomyelin and cholesterol.3,36 In our current study, ASMKO mice at 12 weeks of age (exhibiting significant lysosomal storage pathology within the CNS) were preimmunized with AAV2/1-CBA-null (expressing no transgene) or injected with PBS. As expected, analysis at 12 weeks post-treatment showed the presence of antibodies (1:25,000) against the viral capsid proteins in sera of animals treated with AAV2/1-CBA-Null but not with saline (Figure 7a). At 24 weeks of age, the mice were subjected to intracranial injections of AAV2/1-CBA-hASM into the thalamus of the left hemisphere. Analysis of the sera 6 weeks thereafter showed the presence of antibodies to AAV2/1 in both cohorts; those that had been pretreated with AAV2/1-CBA-Null showed higher levels (>1:200,000) than mice pretreated with PBS (Figure 7a). Measurement of brain hASM levels; however, showed that the presence of pre-existing antibodies to the viral capsids had not affected gene transduction and expression of the lysosomal enzyme (Figure 7b). The levels of hASM were not significantly different between mice that had received a prior injection of a recombinant AAV vector (AAV2/1-CBA-Null) or PBS (Figure 7b). The biodistribution of the enzyme in the brains was also not significantly different in mice that had high or no pre-existing antibodies (Figure 7e–g). Concordant with these results was the observation of a similar extent in reduction of brain sphingomyelin levels in both cohorts of mice (Figure 7c). Mice that had a prior exposure to AAV2/1-CBA-Null also showed a similar extent of reduction in brain cholesterol levels as those that were administered a recombinant virus (AAV1/CBA-hASM) for the first time (Figure 7d,h–k). Collectively, our findings indicate that the presence of circulating antibodies to the viral capsids per se does not significantly affect the efficacy of intracranial injections of AAV2/1-CBA-hASM.

Figure 7.

Figure 7

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Pre-existing anti-AAV2/1 capsid antibodies did not interfere with hASM brain expression and correction of lipid storage. (a) High levels of anti-AAV2/1 capsid antibodies were detected in systemic circulation in ASMKO mice (at 24 weeks of age) that were pretreated with AAV2/1-CBA-Null at 12 weeks of age. Intracranial injection of AAV2/1-hASM (at 24 weeks of age) led to detection of significant anti-AAV2/1 antibodies in serum 6 weeks after surgery (at 30 weeks of age). (b) Analysis showed similar levels of hASM expression in the CNS of AAV2/1-hASM–treated animals in both the saline-pretreated and AAV2/1-CBA-Null–treated cohorts. A similar reduction in (c) sphingomyelin and (d) cholesterol was observed in both the saline-pretreated and AAV2/1-CBA-Null–treated cohorts. ***P < 0.01. Shown are the (e–g) in situ distributions of hASM enzyme and (h–k) cholesterol storage in the brains of (e,h) untreated ASMKO, (f,i) saline-pretreated ASMKO, (g,j) AAV2/1-pretreated ASMKO, and (k) untreated WT mice at 6 weeks postinjection of AAV2/1-hASM. AAV, adeno-associated virus; ASMKO, acid sphingomyelinase knockout; CNS, central nervous system; WT, wild-type.

Discussion

With the emerging interest to deploy recombinant AAV vectors as a gene delivery vector for a number of neurodegenerative diseases, studies were designed to evaluate the relative impact of pre-existing immunity on gene transfer to the CNS.1,2,3,4,5,6,7,8,36,37,38 Although studies had shown that animals exposed previously to AAV precluded subsequent AAV-mediated gene transfer to visceral organs, it was unclear if the “immunoprivileged” CNS would be similarly affected. Studies in rats indicated that for some AAV serotypes, gene delivery to the CNS was blocked when they harbored relatively high neutralizing antibody titers.19,20 As these high titer levels are unlikely to be present in the majority of human patients, we sought to determine the effect of lower titers that are more typically found in the general population, on gene transfer to the mouse CNS. A survey of a small sampling of serum samples from healthy volunteers for circulating antibody titers to AAV2/2 and AAV2/5 established an antibody titer range to model in subsequent mouse studies. Anti-AAV2/2 antibody titers of ≤1:6,400 were observed in 70% of the samples surveyed and anti-AAV2/5 antibody titers of ≤1:1,600 were detected in 71% of the samples analyzed. Together with previous findings of anti-AAV2/8 antibody titers of ≤1:800 in ~89% of samples (same as those used here)21 suggests that modeling a total anti-AAV antibody titer of 1:6,400 in mice might be representative of a large segment of the general population.

As it was unclear whether AAV-mediated gene transfer to the mouse CNS would be negatively affected by pre-existing immunity, the impact of very high circulating anti-AAV antibody titers (>1:400,000) was first assessed. Previous reports had indicated, at least in rats, that relatively high neutralizing antibody titers (i.e., 1:50,000) to AAV2/2, but not to AAV2/5 mitigated gene transfer to the CNS.19 However, contrary to the observations in the rat, pre-existing immunity (at very high total anti-AAV antibody titers) impeded AAV2/5-mediated gene transfer to the mouse CNS. This was perhaps not surprising because mice with very high circulating total anti-AAV2/5 antibody titers also displayed detectable levels of antibodies in brain tissue. The levels of antibodies in the brain tissue were determined to be 0.6% of the levels in systemic circulation. Importantly, the anti-AAV2/5 antibody titer (1:90–1:2,430) found in the mouse brain was within the range reported to be sufficient to impede AAV-mediated gene transfer to the visceral organs.21 The discrepancy between the results observed in mice and that reported in rats could be related to a number of factors. For instance, the method employed to elicit the pre-existing immunity in the mice (intravascular injection of 1.0 × 1011 drp of AAV2/5-βgal at 30 and 60 days before stereotaxic surgery) was likely more immunogenic than that employed in the rat study (combination of subcutaneous and intraperitoneal delivery of 1.0 × 109 drp of the viral vector at 2, 4, and weeks before delivery into the CNS). The degree of immunogenicity of an antigen reportedly can be influenced by the route of administration (subcutaneous = intravenous > intraperitoneal > intramuscular)39 and the dose of antigen (high > low) used.40,41 A higher dose of the test vector was injected into the CNS in the rat study. Recently, we demonstrated that the presence of pre-existing antibodies to AAV2/8 could be partially overcome by increasing the dose of the test vector by tenfold.21 As the rat study employed an approximately tenfold higher dose of the test AAV vector than in our mouse studies, it is possible that the higher dose circumvented the effect of pre-existing immunity to AAV. The contention that vector dose plays a role in determining the efficiency of AAV-mediated gene transfer to the CNS (in the presence of pre-existing anti-AAV antibodies) was further supported by the observation of greater suppression of gene expression in the contralateral (95%) than in the ipsilateral (59%) injected hemispheres of immune-primed mice. This finding was not surprising given that viral exposure in the contralateral hemisphere was likely limited to vector axonal transport from the injected hemisphere—a phenomenon that is largely dependent upon vector dose.6,26 Thus, if a sufficient number of viral particles were neutralized at the injection site (i.e., below the threshold needed to support retrograde transport) then it would be expected that transgene expression within the contralateral side would be significantly reduced. Our findings made in ASMKO mice also support the contention that vector dose plays an important role in modulating the efficiency of AAV-mediated gene transfer to the CNS of immune-primed animals. We observed that intracranial injection of a high dose of AAV2/1-CBA-hASM (similar to that used in the rat study) into ASMKO mice was efficacious despite the presence of high titers (1:25,000) of circulating anti-AAV2/1 antibodies. Collectively, these findings indicate that very high circulating anti-AAV antibodies can result in transfer of sufficient quantities into the CNS to impair gene transfer; however, these untoward effects can be circumvented in part by delivery of higher doses of the viral vector. Importantly, mice with lower levels of pre-existing anti-AAV antibodies (<1:6,400; that are more reflective of those found in the general population) were not effectual in negating gene transfer from a subsequent delivery of recombinant AAV vectors into the CNS.

Studies were also performed to examine whether the site of intracranial injection (ICV versus IP) influenced the extent of neuroinflammation in immune-primed mice. A higher level of immune response might be anticipated from ICV delivery of the viral vector as the ventricular system contains resident dendritic cells, and CSF outflow is largely directed though the lymphatic system.13 Moreover, a greater immune response has been shown associated with ICV injection of recombinant adenoviral vectors than with parenchymal injections.25 Interestingly, no evidence for an enhanced immune response was noted in mice that received ICV injections of the recombinant AAV vectors. Immune-primed mice (with low or high circulating anti-AAV antibody titers) that received ICV administration of the AAV vectors displayed levels of transgene expression within the CNS that were comparable to those of mice that received the vector through IP injection. However, in nonimmune-primed mice, the average antibody titer detected in brain tissue was greater in mice that received IP versus ICV injections. The reason for this observation remains unclear. ICV injection may have elicited a relatively lower immune response because with this approach only cellular components of the ventricular system (i.e., ependymal cells and choroid plexus) are primarily infected whereas with IP injection multiple cells types in a number of brain regions are transduced.38,42

We also investigated what effect pre-existing immunity to AAV had on the relative level of astrocyte, microglial, and T-cell activation within the CNS. Astrocytes have the ability to express the required major histocompatibility complex class II and B7 costimulatory molecules necessary for efficient activation of T cells whereas microglia are capable of secreting a variety of inflammatory mediators including tumor necrosis factor, interleukin (IL)-1β, IL-6, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1, and interferon inducible protein to induce an innate immune response within the CNS.41 In our studies, we found that the neuroinflammatory response observed in immune-primed mice (at 5 and 30 days after vector administration to the CNS) was not significantly enhanced over the response noted in naive mice following intracranial injections of recombinant AAV vectors. Specifically, we observed that the pattern and morphology of astrocyte (GFAP) and microglia (Iba1) staining and changes in histopathology were comparable between mice with pre-existing immunity (regardless of the starting levels of total anti-AAV antibodies) and mice with no prior exposure to AAV. Furthermore, evidence of enhanced T-lymphocyte infiltration (CD3 staining) within the CNS was also not observed in immune-primed mice. It should be noted; however, that mouse models of pre-existing immunity do not stem from natural infection and do not fully recapitulate the human immune response to AAV (e.g., mice do not mimic the CD8+ T-cell response to AAV capsids).43 Nevertheless, these findings suggest that the route of intracranial administration does not influence the efficiency of gene transfer to the CNS of immune-primed mice (with low levels of antibodies) and that re-exposure to recombinant AAV vectors does not generate an enhanced neuroinflammatory response. Collectively these findings are consistent with reports of safe and effective AAV-mediated gene transfer within the CNS of both non-human primates and humans.14,15,16,44,45 We posit that this was likely due, at least in part, to the very limited ability (~0.6%) of circulating antibodies to traverse from the systemic circulation across the blood–brain barrier into the CNS.

Although AAV vectors has been widely used to halt or slow disease course in several animal models of neurodegenerative disease,1,2,3,4,5,6,7,8,36,37,38 no study to date has assessed the efficacy of an AAV-based gene therapy in an immune-primed animal model exhibiting CNS disease. Previously, we had demonstrated that AAV-mediated gene transfer of hASM to the CNS of ASMKO mice, an animal model of NPA disease, is effective in reducing disease-associated sphingomyelin and cholesterol accumulation within the CNS.3,36 In our current experiments, we showed for the first time that similar results could be achieved in ASKMO mice with pre-existing immunity to AAV. Specifically, we demonstrated that immune-primed and AAV naive mice displayed equivalent enzyme distribution within the brain and similar reductions in sphingomyelin and cholesterol following gene delivery of hASM to the CNS. These findings show for the first time that low levels of pre-existing immunity to AAV does not negatively impact the efficacy of an AAV-based therapy in the context of CNS disease.

In summary, we demonstrated that levels of anti-AAV antibodies found typically in the general population does not negatively influence subsequent AAV-mediated gene transfer to the mouse CNS or prime a potentially neurotoxic neuroinflammatory response. Moreover, even in the presence of relatively high (i.e., clinically irrelevant) circulating anti-AAV antibody titers, AAV-mediated gene transfer to the CNS is not completely blocked. Importantly, this impediment could be overcome by the administration of higher doses of the viral vectors. These findings support the continued development of AAV-based therapies for the treatment of neurodegenerative diseases.

Materials and Methods

AAV vectors. Recombinant AAV vectors (AAV2/5-CBA-βgal, AAV2/5-CBA-hIGF-1, AAV2/2-CBA-hIGF-1, AAV2/1-CBA-null, and AAV2/1-CBA-haSM) were produced by triple transfection (using calcium phosphate) of human embryonic kidney carcinoma 293 cells (HEK-293) as previously described.46 Briefly, a plasmid containing the rep gene from serotype 2 and a capsid gene from either serotype 1, 2, or 5 along with a helper adenoviral plasmid (Stratagene, Palo Alto, CA) was used. Virus was collected 72 hours post-transfection and processed on cesium chloride gradients as previously described.47 A contract manufacturing company (Virapur, San Diego, CA) was used for some virus preparations. The cDNA for the hIGF-1 was generated as previously described.4 It encodes the Class 1 IGF-1Ea with a portion of the 5′UTR of IGF-1. AAV2/5-βgal and AAV2/1-hASM constructs were generated as previously reported.26

Animals. Mice were housed under light: dark (12:12 hours) cycle and provided with food and water ad libitum. All procedures were performed using protocols approved by Genzyme's Institutional Animal Care and Use Committee.

Impact of very high circulating anti-AAV2/5 antibodies on AAV2/5-mediated gene transfer to the mouse CNS. Ten week old male B6SJL mice were assigned to one of the following five groups (n = 19/group): (i) AAV naive/sham; (ii) AAV naive/IP; (iii) preimmunized/IP; (iv) AAV naive/ICV; (v) preimmunized/ICV. AAV naive mice received intravascular injections of PBS (100 µl) whereas preimmunized mice were injected with AAV2/5-βgal (1011 infectious particles/injection) using a similar route of delivery. Intravascular injections were performed 60 and 30 days before stereotaxic surgery. Following systemic pre-treatment with either PBS or AAV2/5-CBA-βgal, mice were either immediately killed (n = 8/group) to determine the relationship between anti-AAV antibody titers levels present in serum (at killing) and CNS tissue or subjected to stereotaxic surgery (n = 11/group). Mice that underwent sham stereotaxic surgery were injected with a CSF (3 µl) whereas mice that received either IP (i.e., aimed at the striatum in the right hemisphere) or ICV (i.e., right lateral ventricle) brain injections were injected with AAV2/5-IGF-1 (5 × 109 drps in 3 µl). Mice were killed at either 5 or 30 days after stereotaxic surgery. Mice killed at 5 days after surgery were processed for histological analysis (n = 3/group), whereas mice killed at 30 days were processed for both biochemistry (n = 8/group) and histological (n = 3/group) analysis. Serum was collected from all mice 1 day before stereotaxic surgery and at killing.

Circulating anti-AAV antibody titers following intravenous injection of AAV2/5-βgal at various doses. Ten week old male B6SJL mice received intravascular injections of AAV2/5-CBA-βgal at one of the following doses (n = 5/dose): 1E1, 1E3, 1E5, 1E7, 1E9, and 1E11 drps and were then bled (retro-orbitally) 30 days later to determine circulating anti-AAV antibody titers.

Impact of clinically relevant circulating anti-AAV antibodies on subsequent AAV-mediated gene transfer to the mouse CNS. Ten week old male B6SJL mice were assigned to one of the following seven groups (n = 19/group): (i) AAV naive/sham; (ii) AAV naive/IP AAV2/2-CBA-hIGF-1; (iii) preimmunized/IP AAV2/2-CBA-hIGF-1; (iv) AAV naive/IP AAV2/5-CBA-hIGF-1; (v) preimmunized/IP AAV2/5-CBA-hIGF-1; (vi) AAV naive/ICV AAV2/5-CBA-hIGF-1; (vii) preimmunized/ICV AAV2/5-CBA-hIGF-1. AAV naive mice received intravascular injections of PBS (100 µl) whereas preimmunized mice were injected with Gamunex IVIG as previously reported.28,29 Briefly, mice were injected with 50 mg (in 500 µl) of Gammunex 24 hours before AAV delivery to the CNS. Following systemic pre-treatment with either PBS or Gamunex IVIG mice were either killed (n = 8/group) to determine the relationship between anti-AAV antibody titers levels present in serum (at killing) and CNS tissue or subjected to stereotaxic surgery (n = 11/group) 24 hours later. CNS vector administration (AAV2/2-hIGF-1 or AAV2/5-hIGF-1), serum collection, animal killing, and tissue analysis were completed exactly as performed in experiments in which pre-existing immunity was initiated through systemic exposure to AAV2/5-CBA-βgal.

Impact of pre-existing antibodies on AAV-mediated gene delivery and therapeutic efficacy in a mouse model of neurometabolic disease. Twelve week old male ASMKO mice were assigned to one of the following three groups (n = 12/group): (i) AAV naive/sham; (ii) AAV naive/IP; and (iii) preimmunized/IP. A group of age-matched male wild-type mice (n = 12) were also used as disease controls. AAV naive mice received intravascular injections of PBS (100 µl), whereas preimmunized mice were injected with 3.0 e11 drps of AAV2/1-CBA-null (expressing no transgene). At 24 weeks of age (12 weeks postsystemic pre-treatment) ASMKO mice underwent stereotaxic surgery (1.5 e10 drps of AAV2/1-CBA-hASM into the thalamus of the left hemisphere). Brains were harvested from mice after killing at 30 weeks of age for either biochemical (i.e., hASM protein and sphingomyelin levels; n = 8/group), or histological (hASM immunohistochemistry and filipin staining; n = 4/group) analysis. Serum was collected from mice at 12, 24, and 30 weeks of age.

Stereotaxic surgery. After being anesthetized with isoflurane, mice were unilaterally injected with AAV vectors into either the striatum (A-P: 1.8 from bregma, M-L: −1.3 from bregma, D-V: −2.4 from dura, incisor bar: 0.0), lateral ventricle (A-P: 0.3 from bregma, M-L: −1.0 from bregma, D-V: −2.0 from dura, incisor bar: 0.0) or thalamus (A-P: 0.6 from bregma, M-L: −0.6 from bregma, D-V: −2.2 from dura, incisor bar: 0.0). Vectors were delivered with a 10 µl Hamilton syringe at a rate of 0.5 µl/minute. The final injection volume for each vector was 3 µl/injection. One hour before and 24 hours after surgery mice were given ketoprofen (5 mg/kg; subcutaneously) for analgesia.

Analysis of hIGF-1, hASM, and sphingomyelin levels in brain tissue. Protein was isolated by rapidly dissecting the brain on ice into respective hemispheres and immediately homogenized using TPER reagent (Pierce, Rockford, IL) which included the complete protease inhibitor cocktail (Roche, Palo Alto, CA). Brains from B6SJL male mice injected with AAV vectors encoding hIGF-1 were analyzed for IGF-1 levels as previously described.4 In experiments involving ASMKO mice and wild-type controls, measurement of brain hASM protein and sphingomyelin levels were completed as previously described.48,49

Determination of total anti-AAV2/5 and -AAV2/2 antibody titers. To determine anti-AAV2 and anti-AAV5 titers, ELISA plates (Corning, Oneonta, NY) were coated overnight at 4 °C with 1E9 drp/well of AAV2/2-IGF1 or AAV2/5-hIGF1 in 0.1 mol/l NaHCO3 at pH 9.2. Plates were washed and blocked with 0.5% bovine serum albumin in PBS at a pH of 7.2 for a minimum of 1 hour at 37 °C. After removal of the blocking solution and plate wash, serum samples were diluted twofold serially in duplicate across the plate with a starting dilution of 1:200 in PBS containing 0.5% bovine serum albumin and 0.05% Tween-20 and incubated for 1 hour at 37 °C. Brain samples were processed in a similar manner with a starting dilution of 1:30. Mice were perfused with PBS before brain tissue collection. The entire right hemisphere was homogenized and assayed for total anti-AAV antibody titers. For mouse antibodies, a 1:10,000 dilution of the horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Immunology Consultants Laboratory, Newburg OR) was applied to the plates and incubated for 1 hour at 37 °C. For human antibodies, a 1:5,000 dilution of the horseradish peroxidase-conjugated goat anti-human IgG was used. The ELISA was developed using TMB One Component Microwell Substrate (BioFX Labs, Owings Mills MD) in the dark for 30 minutes. The reaction was stopped with 450 nm Stop Reagent (BioFX Labs) and the plates read in a plate reader (Molecular Devices Spectra Max M2; Molecular Devices, Sunnyvale, CA) at 450 nm. Titers are expressed as the reciprocal of the minimum serum dilution giving an OD450 ≤0.1.

GFAP, Iba1, CD3, H&E, hASM, and filipin staining. Frozen brain sections were stained with either rabbit anti-GFAP antibody to stain astrocytes (1:2,500; DAKO, Glostrup, Germany) or anti-Iba1 antibody to visualize microglia (1:500; WAKO Chemicals USA, Richmond, VA) or anti-CD3 (1:50; R&D Systems, Minneapolis, MN) to stain T lymphocytes. Secondary antibodies used were donkey anti-species–specific antibodies conjugated with FITC or Cy3. Sections were visualized using a Nikon Eclipse E800 fluorescent microscope (Nikon, Melville, NY). Additional sections were processed for H&E, hASM, and filipin staining as previously described.3,26

Statistics. hIGF-1, hASM, sphingomyelin, and filipin levels were analyzed with separate one-way analysis of variances. Follow-up analyses were conducted with Dunnett's post-hoc test where appropriate. All values were considered significant if P < 0.05.

SUPPLEMENTARY MATERIAL Figure S1. Pre-treatment with Gamunex precluded AAV-mediated transgene expression within the mouse liver.

Acknowledgments

We also like to thank Tatyana Taksir, Kuma Misra, Catherine O'Riordan, Anthony Song, Abraham Scaria, Michael Lukason, and the Department of Comparative Medicine at Genzyme for their support and insight on these experiments. All authors are paid employees of Genzyme, a Sanofi Company.

Supplementary Material

Figure S1.

Pre-treatment with Gamunex precluded AAV-mediated transgene expression within the mouse liver.

Click here for additional data file. (282KB, doc)

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Associated Data

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Supplementary Materials

Figure S1.

Pre-treatment with Gamunex precluded AAV-mediated transgene expression within the mouse liver.

Click here for additional data file. (282KB, doc)

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