In the KRAB-based regulation system, TetR-KRAB binds specifically to the sequences in the absence of Dox, thus suppressing the activity of the nearby promoter(s)

In the KRAB-based regulation system, TetR-KRAB binds specifically to the sequences in the absence of Dox, thus suppressing the activity of the nearby promoter(s). models. Our laboratory, in a variety of experimental settings, has evaluated the Dox-regulatable Tet-ON system in rodents and NHP models using the rAAV platform. The general configuration of our vector construct consisted of a single expression cassette flanked by the AAV2 Inverted Terminal Repeat (ITR) sequences and containing the transgene of interest and the chimeric transactivator rtTA. The transgene of interest was placed under the control of the doxycycline-inducible Ptet-1 promoter, in which Tet operator ((TetR) to the C-terminal portion of VP16 of herpes simplex virus (HSV). The rtTA epitope(s) – either originating from one of the two domains or both – recognized by the macaque immune system remains unknown, but if the transactivator domain of the molecule bears the dominant epitopes, then using a less antigenic transactivator protein would potentially be beneficial to support long-term transgene regulation. By fusing the KRAB domain of the human zinc-finger protein Kox1 to the DNA binding domain of the Tet repressor, Deuschle generated a Dox-sensitive transrepressor called TetR-KRAB [20]. Krppel associated box (KRAB) is an approximately 75-amino acid transcriptional repression domain found in many mammalian zinc finger-containing proteins, which can suppress, in an orientation-independent manner, polymerase I, II and III-mediated transcription within a distance of up to 3 kb from its DNA binding site, presumably by triggering the formation of heterochromatin [20], [21], [22]. An understanding of the mechanism of action of KRAB has been achieved through the identification and characterization of KRAB-associated protein 1 (KAP1), believed to represent its universal corepressor [23]. In Rabbit polyclonal to SHP-1.The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. the KRAB-based regulation system, TetR-KRAB binds specifically to the sequences in the absence of Dox, thus suppressing the activity of the nearby promoter(s). In contrast, upon Dox administration, TetR-KRAB is sequestered from sequences, allowing transgene expression after KRAB-mediated transcription repression is lifted [20], [21], [22]. Using the lentiviral vector platform, the system allowed concise gene expression switch even when low amounts of Dox were added to the culture [24] and over several induction cycles [25] and in a mouse model, using either integration-deficient lentiviral vectors or rAAV-based vectors. Recently, another study demonstrated the functionality of the TetR-KRAB repressor-based system after IM delivery of a rAAV2/8 vector in the mouse [29]. In this study, we evaluated the ability of the TetR-KRAB system to mediate concise and reproducible transgene transcriptional regulation after subretinal rAAV delivery in rats. Because the efficiency and immunogenicity of the TetR-KRAB based-system has not Temsirolimus (Torisel) been explored in higher species, we also evaluated the system after IM delivery Temsirolimus (Torisel) in mice a macaque model. Because the human and putative macaque Kox1 nucleotide sequences are more than 95% homologous, we hypothesized that exchanging the HSV VP16 domain with the human KRAB one may result in less immunotoxicity than when the rtTA transactivator is expressed from the macaque skeletal muscle. Results rAAV.TetR-KRAB/GFP in the rat retina results in long term Dox-mediated transgene regulation For retinal gene transfer, we used rAAV vectors in which the TetR-KRAB expression was driven by the ubiquitous CAG promoter and the reporter gene was under the control of the construct, the CAG promoter, driving TetR-KRAB expression, was located 2.5 kB from the sequences. Because the TetR-KRAB protein has been demonstrated to be capable of inhibiting all promoters within at least 3 kB [20] and the construct would therefore inhibit the CAG promoter, we generated the construct, where the two expression cassettes are cloned in a manner where both promoters are at the opposite ends at a distance of 4 kB. Using these two expression cassettes, we evaluated two different rAAV5 vectors: rAAV5.d2GFP.KRAB and rAAV5.d2GFP.KRAB fluorescence imaging to directly monitor the appearance of GFP expression. A weak GFP signal, representing the background levels of protein expression in the absence of induction, appeared in all retinas within 2 months following vector injection ( Figure 1B and 1C , before induction) compared with non-injected rats (data not shown). Open in a separate window Figure 1 rAAV.TetR-KRAB/d2GFP in the rat retina results in Dox-mediated transgene regulation.(A) Vector design: Vectors encode the destabilized GFP (d2GFP) under the control of the Temsirolimus (Torisel) and (C) rAAV5.d2GFP.KRAB the drinking water, the GFP.