Abstract
The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl β-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.
Highlights
The field of gene therapy has seen many significant advances over the past decade[1,2,3]
Most transgene regulatory systems are based upon the classical bacterial operons, where a regulating protein is constitutively produced by one promoter to modulate transcription of a second promoter expressing a functional gene[6]
Regardless of how the regulator is controlled, the fundamental problems that have plagued all of these regulatory systems is that effector molecules can produce unwanted side effects, the regulatory circuitry exhibits a high basal level of gene expression with only a modest dynamic range, and many of these systems are too large to fit within the packaging constraints of a single viral vector such as a recombinant adeno-associated virus
Summary
We have established that an autogenously regulated expression system exhibits similar steady state induction profiles compared to the classical operon model system Both regulatory systems successfully control transgene expression in a variety of cell types both in vitro and in vivo, the autogenous system may be more useful for gene therapy. An autogenously regulated system satisfies these objectives and, as we have shown here, is comparable in kinetics and dynamic range to traditional constitutively expressed transgene regulatory systems without the need for promoter balancing or empiric tuning for different applications These data establish the proof-of-concept of using autogeny and the lac repressor to control transgenes in AAV-mediated gene therapeutic applications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
