Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus

Abstract

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.

FIG. 1.

Structure of the AAVcm-ET(CAG) vector. pA, bidirectional SV40 polyadenylation signal; CAG, CAG promoter (22). BamHI restriction sites and resulting fragments are indicated. The CAG promoter was replaced by the retroviral MFG long terminal repeat (7) in the AAVcm-ET(LTR) vector (not represented). ITR, inverted terminal repeat; Epo, erythropoietin cDNA from Macaca fascicularis.

FIG. 3.

Epo and reticulocyte levels, obtained from the last induction peak from Mac 8 (Fig. 2a) and determined on a daily basis upon DOX i.v. administration for 5 days (dotted area) and subsequent withdrawal.

FIG. 2.

Epo (a and b) and hematocrit (dotted lines) and reticulocyte (solid lines) (c and d) levels in representative animals Mac 6 and Mac 8. Solid arrows (a and b), DOX administration. Animals were occasionally bled (open arrows [c and d]). Ab (rtTA), detection of anti-rtTA antibodies in successive serum samples; c+, positive control consisting in a commercial specific MAb against rtTA; c−, negative control obtained from sera prior to rAAV administration.

FIG. 4.

Muscle biopsy sample obtained from Mac 6 6 months after rAAV injection. Shown is a phenotypic characterization of the cellular infiltrates. (a) Hematoxylin and eosin staining. Magnification, ×100. (b) Anti-CD68-AP. Magnification, ×200. (c) Anti-CD8-horseradish peroxidase. Magnification, ×300. (d) Anti-CD4-AP. Magnification, ×400.

FIG. 5.

Cytokine production can be measured at the single-cell level by using the ELISPOT technique, allowing calculation of T-cell frequencies. Shown is an IFN-γ ELISPOT assay using autologous LNMCs from Mac 6 and Mac 8, obtained from inguinal draining lymph nodes in the presence of autologous DCs expressing the rtTA antigen (LNMC/DC-rtTA [a and c]) or GFP (LNMC/DC-GFP [b]). Herpesvirus papio-transformed B lymphoblastoid cell lines (papio B) were derived from PBMCs of Mac 6 and Mac 8 and were then infected with either vaccinia virus vtetR (papio B + vtetR) or control wild-type vaccinia virus (papio B + vWR). Spot quantification is described in Materials and Methods. Eff, effector cells.

FIG. 6.

Southern blot analysis of high-molecular-weight DNA extracted from muscles biopsy samples. (a) rtTA probe; (b) tetO-CMV probe. Muscle samples were obtained at +1, +6, and +12 months for Mac 6 (lanes 1 to 3) and at +15 months for Mac 7 (lane 5) and Mac 8 (lanes 4 and 6). Lane 7 corresponds to ∼10⁹ single-stranded vector genomes. Reference copy numbers correspond to high-molecular-weight DNA extracted from normal macaque muscle and run with plasmid DNA corresponding to 0.01 copy (0.25 pg) up to 1 copy (25 pg) of pAAVcm-ET(CAG). Open arrowheads, 1.4-kb single-stranded vector DNA input; filled arrowheads, 1-kb band (a, lanes 1 and 4) and ∼1.8-kb band compatible with a head-to-tail concatemer (b). Quantification of vector signals relative to the reference copy number signals was done on a phosphorimager.

FIG. 7.

Epo and rtTA RNA expression from rAAV-transduced muscles. Total RNA was extracted and processed as described in Materials and Methods. Samples were separated by electrophoresis, subsequently blotted under alkaline conditions (except for the HPRT control), and then hybridized with [³²P]dCTP random-primed labeled Epo (a) and rtTA (b) probes. (c) Control of RNA extraction. Lack of a DNA contaminant before reverse transcription is shown by the RT− samples. C⁺, control PCR on 50 pg of AAV vector plasmid [pAAVcm-ET(CAG)]; NI. muscle, noninjected muscle; I. muscle, rAAV-injected muscle; M, marker.