Lack of humoral immune response to the tetracycline (Tet) activator in rats injected intracranially with Tet-off rAAV vectors

Abstract

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression.

Figure 1. Expression of hAADC and hGDNF was tightly regulated by dox in rats that received an intrastriatal injection of either rAAV2S3hAADC or rAAV2S3hGDNF

The representative immunohistochemical images show high hAADC expression in the striatum of rats maintained on the normal water (A) and low hAADC expression in rats maintained on the water containing dox (B). (C). Relative mRNA levels of hGDNF in striatum evaluated by qRT-PCR. The rats of rAAVS3-hGDNF in “on” state showed the highest expressional levels of hGDNF, which was significantly higher than the “hGDNF off” group (P<0.001). (D). hGDNF protein in the striatum measured by ELISA. The rats injected with rAAVS3-hGDNF maintained on normal water showed the highest levels of hGDNF protein on the ipsilateral side, which was significantly higher than that in the “hGDNF off” group (P<0.001).

Figure 2. GST-tTA fusion protein purification

(A). Schematic map of the molecular mass of GST-tTA fusion protein. tTA is comprised of tet repressor protein (tetR) and a transactivator domain, VP16. (B). Upper panel: bacterial pellets transformed with pGEX-tTA were subjected to 4-15% SDS–PAGE followed by staining with Coomassie blue. Lane 1, culture lysate without IPTG induction. Lane 2, culture lysate with 1hr IPTG induction. Lane 3, cell lysate were frozen at -80°C overnight followed by thaw in PBS. Lane 4, fresh cell lysate dissolved in PBS. Lane 5, purified GST-tTA using freeze-thawed pellets. The top band is GST-tTA and the bottom band is GST-tetR, showing that the majority of the purified fusion proteins are broken down. However, the cleavage was significantly decreased using fresh bacterial pellets. The majority of the purified fusion proteins are GST-tTA (Lane 6). Bottom panel: western blot results detected by anti-tetR antibodies.

Figure 3. Positive controls to verify the anti-tetR ELISA

The positive controls included sera from rats that received intradermal injection of rAAV2S3hrGFP or an Ad containing either tet-off (AdBIE5/SV40) or tet-on (Ad-tet-on). The sera were collected at 4 weeks after injection. No detectable antibodies were observed in sera from any of the rats injected with rAAV2S3hrGFP. However, sera from 1 out of 3 rats that received AdBIE5/SV40 vector showed a detectable level of anti-tTA (P<0.01). The peak level was observed in the sera from rats that were injected intradermally with Ad-tet-on (P<0.001).

Figure 4. Lack of humoral immune response to tTA in rats that received an injection of rAAV harboring tTA into the striatum

No detectable antibodies against tTA were observed in plasma from rats that were intrastriatally injected with either 5 μl of 1.6×10e13 vg/ml of rAAV2S3hAADC or 4 μl of 7.2×10¹³ vg/ml of rAAV2S3hGDNF (P>0.05). Plasma of rats injected with AAVhAADC was collected 9 weeks post injection (n=21), and the plasma of rats injected with AAVhGDNF was collected 5 weeks post injection (n=9). Pre-immune plasma and plasma from rats injected intrastriatally with saline were used as negative controls (n=12). (B) At 9 weeks post injection, microglial activity was assessed by immunochemical staining of Iba1 in rats injected with AAVS3-hAADC (n=3) and saline (n=3).