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.Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer in skeletal muscle of mice (36), dogs (13), nonhuman primates (8, 37), and hemophilia patients (16) is well tolerated and is associated with long-term expression. As such, it becomes possible to evaluate strategies which allow long-term transgene regulation; such strategies are likely to be required for therapeutic applications and in some instances for safety reasons. A rather limited number of clinically translatable regulatory systems are available. They all have in common the use of chimeric transactivators, the activity of which is controlled by drugs including tetracycline (11), mifepristone (35), ecdysone (23), and rapamycin (25).The rapamycin-regulatable system uses rapamycin or its analog to bring together the functional units of bipartite chimeric transcription factor ZFHD1/FKBP-FRAP/p65 (25). Their corresponding cDNAs have been included in an rAAV vector and injected intramuscularly (i.m.) in macaques along with a second rAAV harboring the erythropoietin (Epo) cDNA under the control of a ZFHD1-dependent promoter. This resulted in long-term regulation of Epo secretion in mice and regulation for up to 3 months in one rhesus macaque out of three (37).The repressor of the Tn10 tetracycline resistance operon of Escherichia coli (tetR) recognizes its operator (tetO) ...

Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives

“…46 The tetracycline system has also proved to be efficient and reliable in controlling transgene expression in vivo following intramuscular injection of rAAV carrying the tetracycline transactivator and Epo cDNA in mice 47,48 and in primates. 49 In the retina, tetracycline-controlled expression of GFP in RPE and photoreceptor cells has been demonstrated in rats following subretinal injection of rAAV-2/2 vector. 50 However, restricting transduction to the neuroretina, and more specifically to ganglion cells, following intravitreal (not subretinal) injection, would be a more attractive approach for neurotrophic factor gene transfer as it should allow transduction over the entire retina.…”

Complement Is an Essential Component of the Immune Response to Adeno-Associated Virus Vectors

“…The inability of AAV vectors to efficiently transduce or activate antigen-presenting cells may account for their decreased immunogenicity (74). However, AAV vectors can induce cellular and humoral responses to the transgene product (15,21,22,41,43,49,71) and AAV-mediated gene therapy leads to the development of antibodies against the vector capsid, confirming that a significant interaction with the immune system exists (9,28,55). Anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo gene therapy and can prevent vector readministration (13,52).…”

Application des techniques de thérapie génique aux maladies ostéo-articulaires

“…Chez le singe [33] et la souris [34], ces constructions permettent d'obtenir un niveau d'expression basale du transgène négligeable en l'absence de doxycycline, ainsi qu'un contrôle strict de l'expression du transgène sur plusieurs années. Cependant, le potentiel immunogène de ce système fait l'objet de résultats contradictoires [35,36]. Afin de s'affranchir de ce problème, le système inductible par la rapamycine (Rp) n'utilise que des éléments d'origine humaine [37,38].…”