Immunological aspects of recombinant adeno-associated virus delivery to the mammalian brain

Abstract

Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene delivery into the central nervous system (CNS). However, host inflammatory and immune responses may play a critical role in limiting the use of rAAV vectors for gene therapy and functional genomic studies in vivo. Here, we evaluated the effect of repeated injections of five rAAV vectors expressing different genetic sequences (coding or noncoding) in a range of combinations into the rat brain. Specifically, we wished to determine whether a specific immune or inflammatory response appeared in response to the vector and/or the transgene protein after repeated injections under conditions of mannitol coinjection. We show that readministration of the same rAAV to the CNS is possible if the interval between the first and second injection is more than 4 weeks. Furthermore, our data demonstrate that rAAV vectors carrying different genetic sequences can be administered at intervals of 2 weeks. Our data therefore suggest that the AAV capsid structure is altered by the vector genetic sequence, such that secondary structures of the single-stranded genome have an impact on the antigenicity of the virus. This study provides guidelines for more rational design of gene transfer studies in the rodent brain and, in addition, suggests the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.

FIG. 1.

AAV vectors. AAV ITRs (TR), coding regions (Luc, TH, and GFP), promoters (NSE and CBA), regulatory elements (WPRE), and polyadenylation sequences (pA) are indicated. All vectors shown contain AAV2 ITR.

FIG. 2.

Repeated rAAV injections into rat brain at different time points and effects of a host immune response on transgene expression. The luciferase-expressing AAV/NSE-Luc vector was injected into rat striatum, and a second rAAV injection followed after 2 weeks (A), 4 weeks (B), or 3 months (C). The control groups of rats received a single injection at the second time point only. All rAAVs were coinjected with mannitol, and luciferase enzyme activity was determined 4 weeks after the second injection. Note the 10- and 5-fold decreases in luciferase activity in the groups that received the second rAAV injection after 2 and 4 weeks, respectively (A and B), suggesting the presence of a host humoral immune response that could have an impact on the transgene expression levels. In the groups that received the second rAAV administration 3 months (C) after the first injection, transgene activity was as high as in the control groups, indicating a lack or minor effect of a host immune response. The level of luciferase expression was dependent on the time period between the first and second injection (n = 4 to 6 per group; total n = 41; F_{(7, 33)} = 7.703; P < 0.0001). Error bars, SEM. (D to F) Fluorescent microscopic images showing autofluorescence of EGFP (green) of representative striatal sections after repeated administration of AAV/NSE-GFP virus 3 months apart. (D and E) Transgene expression at the first injection site (3-month transgene expression period) (D) and at the second injection site (1-month transgene expression period) (E). (F) EGFP expression in a section of a control brain that received a single injection at the second time point only (1-month transgene expression period). Note the homogenous and constantly high expression level in all sections. Scale bar, 150 μm.

FIG. 3.

Repeated rAAV injections into rat striatum after 2 weeks and effects of a host immune response on transgene expression. Rats received a first injection of AAV/NSE-Luc vector (A), AAV/NSE-TH (B) or AAV/CBA-Luc (C) followed by a second injection of AAV/NSE-Luc after 2 weeks. The control groups received only a single injection of AAV/NSE-Luc at the second time point. All rAAVs were coinjected with mannitol, and luciferase enzyme activity was determined 4 weeks after the second injection. Note the 10-fold reduction in luciferase enzyme activity if the same virus was used for the second injection (A) and that no decrease in luciferase enzyme activity was observed if AAV/NSE-TH (B) or AAV/CBA-Luc (C) was injected prior to the AAV/NSE-Luc vector. The level of AAV/NSE-Luc expression was dependent on the second virus injected (n = 4 to 6 per group; total n = 20; F_{(3, 16)} = 15.319; P < 0.0001). Error bars, SEM.

FIG. 4.

Injecting rAAV5 and 2 weeks later rAAV2 did not change GFP transgene expression levels. Rats received a first injection of AAV5/EGFP followed by a second injection of AAV/NSE-GFP after 2 weeks. The control group received only a single injection of AAV/NSE-GFP at the second time point. The fluorescent microscopy images show autofluorescence of EGFP of representative striatal sections 4 weeks after the second virus administration. Note the homogenous and constantly high expression level in all sections, indicating that virus spread and transgene expression was not effected after injecting rAAV5 followed by rAAV2 after 2 weeks. Scale bar, 300 μm.

FIG. 5.

Immunoneutralization assay showing effects of rAAVs differing in their transgenes on neutralizing antibody formation and transgene expression. The sera from three animals that received AAV/CBA-Luc injections in the brain (A and B) or from three naive animals (C and D) obtained at 2 weeks after injection were pooled, and 100 μl was added to 1 μl of virus (AAV/CBA-Luc [A and C] or AAV/CBA-GFP [B and D]) and directly incubated on HEK293 cells for transduction. Immunocytochemistry was performed after 2 to 3 days to determine transgene expression using antibodies against the Luc (A and C) or GFP transgene (B and D). Note that the AAV/CBA-Luc serum from rats injected into the brain reduced AAV/CBA-Luc expression (A) but not AAV/CBA-GFP expression (B). Incubation of serum from naive rats with AAV/CBA-Luc (C) or AAV/CBA-GFP (D) virus had no effect on the transgene expression. These results indicate the presence of AAV/CBA-Luc-specific antibodies in the serum of AAV/CBA-Luc brain-injected rats. Scale bar, 300 μm.

FIG. 6.

Lack of significant tissue damage and inflammatory responses after rAAV administrations. Representative sections from each group were immunohistochemically analyzed using antibodies against isolectin B4 (left column), T-cell helper/macrophage CD-4 (middle column), and suppressor or cytotoxic cell CD-8 (right column). Fluorescence microscopy images of sections showed no detectable cytotoxic tissue damage after repeated injections (row A) and no difference between the groups (row B) (control animals received a single injection of rAAV). Scale bar, 300 μm.