Insulators coupled to a minimal bidirectional tet cassette for tight regulation of raav-mediated gene transfer in the mammalian brain

ABSTRACT Recombinant AAV is increasingly becoming the vector of choice for many gene therapy applications in the CNS, due to its lack of toxicity and high level of sustained expression. With

recent improvements in the generation of pure, high titer vector stocks, the regulation of gene expression is now a key issue for successful translation of gene therapy-based treatments to

the clinic. The level of the transgene protein may need to be maintained within a narrow therapeutic window for the successful treatment of human disease. The doxycycline responsive system

directs a dose-responsive, tightly regulated level of gene expression and has been used successfully in transgenic mouse models. Here, we have optimized an autoregulatory, bidirectional

doxycyline responsive cassette specifically for use in rAAV. We minimized the size of the cassette and decreased the basal leakiness of the system, leading to tight regulation in the rat

brain. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to

this journal Receive 6 print issues and online access $259.00 per year only $43.17 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy

now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer

support SIMILAR CONTENT BEING VIEWED BY OTHERS SPATIAL GENOMICS OF AAV VECTORS REVEALS MECHANISM OF TRANSCRIPTIONAL CROSSTALK THAT ENABLES TARGETED DELIVERY OF LARGE GENETIC CARGO Article

Open access 20 March 2025 ENGINEERED COMPACT PAN-NEURONAL PROMOTER FROM ALPHAHERPESVIRUS LAP2 ENHANCES TARGET GENE EXPRESSION IN THE MOUSE BRAIN AND REDUCES TROPISM IN THE LIVER Article Open

access 27 November 2023 A COMPARISON OF AAV-VECTOR PRODUCTION METHODS FOR GENE THERAPY AND PRECLINICAL ASSESSMENT Article Open access 09 December 2020 REFERENCES * Reed Clark K _et al_.

Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses _Hum Gene Ther_ 1999 10: 1031–1039 Article  Google

Scholar  * Monahan PE, Samulski RJ . AAV vectors: is clinical success on the horizon? _Gene Therapy_ 2000 7: 24–30 Article  CAS  PubMed  Google Scholar  * Constantini LC, Bakowska JC,

Breakfield XO, Isacson O . Gene therapy in the CNS _Gene Therapy_ 2000 7: 93–109 Article  Google Scholar  * Navarro V _et al_. Efficient gene transfer and long term expression in neurons

using a recombinant adenovirus with a neuron specific promoter _Gene Therapy_ 1999 6: 1884–1892 Article  CAS  PubMed  Google Scholar  * Peel AL, Klein RL . AAV vectors: activity and

applications in the CNS _J Neurosci Meth_ 2000 98: 85–104 Article  Google Scholar  * Bjorklund A _et al_. Towards a neuroprotective gene therapy for Parkinson's disease: use of

adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model _Brain Res_ 2000 886: 82–98 Article  CAS  PubMed  Google Scholar  *

Shen Y _et al_. Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase aromatic-L-amino-acid decarboxylase and GTP cyclohydrolase I for gene therapy of

Parkinson's disease _Hum Gene Ther_ 2000 11: 1509–1519 Article  CAS  PubMed  Google Scholar  * During MJ _et al_. In vivo expression of the therapeutic human genes for dopamine

production in the caudates of MPTP-treated monkeys using an AAV vector _Gene Therapy_ 1998 5: 820–827 Article  CAS  PubMed  Google Scholar  * Rudich SM _et al_. Dose response to a single

intramuscular injection of recombinant adeno-associated virus-erythropoetin in monkeys _J Surg Res_ 2000 90: 102–108 Article  CAS  PubMed  Google Scholar  * Gossen M, Bujard H . Tight

control of gene expression in mammalian cells by tetracycline-responsive promoters _Proc Natl Acad Sci USA_ 1992 89: 5547–5551 Article  CAS  PubMed  PubMed Central  Google Scholar  * Rivera

VM _et al_. A humanized system for pharmacological control of gene expression _Nat Med_ 1996 2: 1028–1032 Article  CAS  PubMed  Google Scholar  * Strathdee CA, McLeod MR, Hall JR . Efficient

control of tetracycline-responsive gene expression from an autoregulated bidirectional expression vector _Gene_ 1999 229: 21–29 Article  CAS  PubMed  Google Scholar  * Loeb JE _et al_.

Enhanced expression of transgenes from adeno-associated vectors with the woodchuck post-transcriptional regulatory element: importance for gene therapy _Hum Gene Ther_ 1999 10: 2295–2305

Article  CAS  PubMed  Google Scholar  * Flotte TR _et al_. Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter _J Biol Chem_

1993 268: 3781–3790 CAS  PubMed  Google Scholar  * Bell AC, West AG, Felsenfeld G . The protein CTCF is required for the enhancer blocking activity of vertebrate insulators _Cell_ 1999 98:

387–396 Article  CAS  PubMed  Google Scholar  * Gallia GL, Khalili K . Evaluation of an autoregulatory tetracycline regulated system _Oncogene_ 1998 16: 1879–1884 Article  CAS  PubMed 

Google Scholar  * Baron U, Gossen M, Bujard H . Tetracycline-controlled transcription in eukaryotes: novel transactivator with graded transactivation potential _Nucleic Acids Res_ 1997 25:

2723–2729 Article  CAS  PubMed  PubMed Central  Google Scholar  * Baron U, Gossen M, Bujard H . Tetracycline-controlled transcription in eukaryotes: novel transactivator with graded

transactivation potential * Mastakov MY _et al_. Combined injection of rAAV with mannitol enhances gene expression in the rat brain _Mol Ther_ 2001 3: 225–232 Article  CAS  PubMed  Google

Scholar  * Haberman RP, McCown TJ, Samulski RJ . Inducible long-term expression in brain with adeno-associated virus gene transfer _Gene Therapy_ 1998 5: 1604–1611 Article  CAS  PubMed 

Google Scholar  * Bohl D, Salvetti A, Moullier P, Heard JM . Control of erythropoietin by doxycycline in mice after intramuscular injection of adeno-associated vector _Blood_ 1998 92:

1512–1517 CAS  PubMed  Google Scholar  * Freundlieb S, Schirra-Mueller C, Bujard H . A tetracycline controlled activation/repression system with increased potential in gene transfer into

mammalian cells _J Gen Med_ 1999 1: 4–12 Article  CAS  Google Scholar  * Nakai H, Storm TA, Kay MA . Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular

joining of two complementary vectors _Nat Biotechnol_ 2000 18: 527–532 Article  CAS  PubMed  Google Scholar  * Duan D, Yue Y, Yan Z, Engelhardt JF . A new dual-vector approach to enhance

recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation _Nat Med_ 2000 6: 595–598 Article  CAS  PubMed  Google Scholar  * Sun L, Li J, Xiao X .

Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerisation _Nat Med_ 2000 6: 599–602 Article  CAS  PubMed  Google Scholar  * Kistner A _et al_.

Doxycycline-mediated quantitative and tissue specific control of gene expression in transgenic mice _Proc Natl Acad Sci USA_ 1996 93: 10933–10938 Article  CAS  PubMed  PubMed Central  Google

Scholar  Download references ACKNOWLEDGEMENTS We thank R Bland, D Young and T Lawlor for critical reading of the manuscript. We are grateful to C Strathdee for providing the pBIG3r plasmid.

This work was supported by the New Economy Research Fund, New Zealand Health Research Council, the National Institutes of Health and the Jefferson Faculty Foundation. AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Division of Molecular Medicine, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand HL Fitzsimons, JM McKenzie & MJ During *

CNS Gene Therapy Center, Jefferson Medical College, Philadelphia, PA, USA MJ During Authors * HL Fitzsimons View author publications You can also search for this author inPubMed Google

Scholar * JM McKenzie View author publications You can also search for this author inPubMed Google Scholar * MJ During View author publications You can also search for this author inPubMed 

Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Fitzsimons, H., McKenzie, J. & During, M. Insulators coupled to a minimal

bidirectional tet cassette for tight regulation of rAAV-mediated gene transfer in the mammalian brain. _Gene Ther_ 8, 1675–1681 (2001). https://doi.org/10.1038/sj.gt.3301582 Download

citation * Received: 06 April 2001 * Accepted: 13 August 2001 * Published: 17 December 2001 * Issue Date: 01 November 2001 * DOI: https://doi.org/10.1038/sj.gt.3301582 SHARE THIS ARTICLE

Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided

by the Springer Nature SharedIt content-sharing initiative KEYWORDS * rAAV * gene therapy * tetracycline transactivator * autoregulatory * CNS * insulators