Increased utility in the CNS of a powerful neuron‐specific tetracycline‐regulatable adenoviral system developed using a post‐transcriptional enhancer

Bantounas

et al. 2008

Nucleic Acids Research

206

207

MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel intermediates in the pathways controlling the development of specific neural cell populations.

Section: Methodsmentioning

confidence: 99%

Twist-1 regulates the miR-199a/214 cluster during development

Bantounas

et al. 2008

Nucleic Acids Research

206

207

“…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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…Regulatable switches have been previously engineered into several vector systems, i.e., lentivirus (13,23,38,59), retrovirus (19), adeno-associated virus (10,11,20,27,33,40,44), firstgeneration adenovirus (11,27,33,44,47,48), herpesvirus (18), and high-capacity adenoviral vectors (5,6,45,57,60,64). Wang et al determined that an inducible system within a high-capacity adenovirus (HC-Ad) led to prolonged periods of regulatable transgene expression in the liver (60).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Xiong

Goverdhana

Sciascia

et al. 2006

J Virol

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...

“…Such systems can enable the production of vectors that contain toxic inserts, acting specifically and at high levels in the “On” state, and show dose-responsive expression allowing varied levels of gene expression. Subsequent studies have combined this system with cell-specific promoters to drive neuron-specific and glial-specific regulatable transgene expression (Glover et al 2002, 2003; Lee et al 2005; Ralph et al 2000). The well-characterised tetracycline-based regulatable systems have also been utilised in lentiviral vector design (Kafri et al 2000) and AAV-based studies (Stieger et al 2009).…”

Section: Targeted Gene Transfer and Regulatable Expressionmentioning

confidence: 99%

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

2009

Self Cite

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.