Year: 2006
Type: article
Abstract: A number of tetracycline-regulated systems constructed in rAAV vectors were evaluated to control in vivo expression of glial cell line- derived neurotrophic factor (GDNF) in the rat neostriatum. The first system involved two vectors: rAAV construct contained tetracycline (tet) -inducible promoter driving expression of GDNF (tet-GDNF), and the other was a bicistronic vector encoding the reverse transactivator and silencer for the tet-regulated system (rtTA/ tTS). The second vector system incorporated all the elements required for the regulated expression of GDNF in one construct. This arrangement contained a mutated rtTA (rtTA2s-M2), a bidirectional tet-induceable autoregulatory promoter controlling the expression of rtTA2s-M2 and GDNF, in AAV2 inverted terminal repeats (TR2:M2-GDNF). The third construct system was identical to the later vector but contained AAV5 TR (TR5:M2-GDNF). As much as 5.4 fold induction of GDNF over un-induced levels could be achieved in vivo, using the dual vector system. However, measurable background expression was observed in the un-induced state. Un-induced GDNF expression could be reduced to near background levels by increasing the ratio of the reverse transactivator-repressor vector to the tet-GDNF. On the other hand the bidirectional constructs showed high expression of GDNF in the uninduced state, but the TR5 seemed to reduce the expression observed in the uninduced state with the TR2 bidirectional construct. We conclude that a dual vector system might be more efficient at regulating gene expression in the context of rAAV vectors. Based on these results we are developing improved versions of the two vector system to limit uninduced transgene expression and to increase the levels of induction in the “on” state. Dr. Muzyczka is an inventor on patents related to recombinant Adeno-associated virus technology and owns equity in a gene therapy company that is commercializing AAV for gene therapy applications. Dr. Mandel and Dr. Burger are inventors on patents related to recombinant Adeno-associated virus technology.
Source: Molecular Therapy
Institution: University of Florida
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W2085703649