Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses

Abstract

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding ␤-galactosidase (␤-gal) driven by a TetOn system containing the rtTAS s M2 transactivator and the tTS Kid repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-␤-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-␤-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-␤-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-␤-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.The capacity to tightly and effectively turn "on" or switch "off" the expression of a therapeutic gene is critical to achieve successful short-and long-term therapeutic benefits in clinical gene therapy. An inducible system will allow the turning "off" of the therapeutic gene during disease remission or if toxic side effects arise. It will also allow the gene to be turned "on" during exacerbation periods of the disease. Four main regulatory systems are currently available and include the tetracycline-, the progesterone antagonist RU486 (9, 61)-, the insect hormone ecdysone (24)-, and the rapamycin (FK506)-dependent systems (17, 42). We have chosen to use the tetracycline (Tet)-dependent inducible system for transgene expression regulation in the central nervous system (CNS), since the inducers are nontoxic, cross the blood-brain barrier, and provide tight regulation within adenovirus (19,21,22,40,47,48,62).The original tetracycline (Tet)-regulated system is constitutively active, but in the presence of the tetracycline analog, doxycycline (Dox), gene expression is switched "off" and therefore is known as the "tet off" variant (20,27). A mutant tetracycline-dependent transactivator (rtTA) was found to become active only in the presence of Dox (15). The rtTA system is thus called "TetOn," si...

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“…We also constructed a control vector expressing mhIL-4 and mhIL-13 (without the PE) under the control of the tetracycline-responsive (TRE) promoter. The TetON system that regulates the activity of the bidirectional TRE promoter consists of the tetracycline-controlled transactivator rtTA2S-M2, which activates the TRE promoter in the presence of the inducer doxycycline (Dox), and the Tet repressor tTS kid , which inhibits expression in the absence of Dox, inhibiting the leakiness of the system (23,24). The mCMV promoter drives constitutive expression of TetON.…”

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“…While, transgene expression from a first-generation Ad vector was completely eliminated following peripheral immune priming, HD-Ad vectors produced sustained transgene expression in the rat brain . Even in the presence of anti-HAd immunity, an HD-Ad system resulted in sustained and regulatable transgene expression in the brain [Xiong et al 2006]. It may obviate the need to screen patients for pre-existing vector immunity especially for gene delivery to the brain.…”