Gene regulation systems for gene therapy applications in the central nervous system

Abstract

Substantial progress has been made in the development of novel gene therapy strategies for central nervous system (CNS) disorders in recent years. However, unregulated transgene expression is a significant issue limiting human applications due to the potential side effects from excessive levels of transgenic protein that indiscriminately affect both diseased and nondiseased cells. Gene regulation systems are a tool by which tight tissue-specific and temporal regulation of transgene expression may be achieved. This review covers the features of ideal regulatory systems and summarises the mechanics of current exogenous and endogenous gene regulation systems and their utility in the CNS.

Figure 1

Mechanics of tetracycline regulated systems. A constitutively active promoter drives expression of tetracycline transactivators (tTAs) or reverse tetracycline transactivators (rtTAs). (a) Tet-Off system. tTAs are able to bind to the tet-operator sequence (tetO) in the absence, but not in the presence of doxycycline (dox) to drive transgene expression. (b) Tet-On system. rtTAs are able to bind to the TRE in the presence, but not in the absence of dox to drive transgene expression. tetR: tetracycline repressor; VP16: viral protein 16; CMV: cytomegalovirus.

Figure 2

Different configurations the tetracycline system has been incorporated into viral vectors for use in the brain. (1) Two right-facing cistrons direct transactivator and transgene expression in a single vector [–7]. (2) One bidirectional promoter driving expression of the transgene and transactivators in a single construct [, , –10]. (3) Two vectors are used: one bidirectional construct directing expression of two transgenes and one driving expression of transactivators [11, 12]. ITR: inverted terminal repeats; PolyA: polyadenylation signal; TetO: tetracycline operator sequence; CMV: cytomegalovirus; tTA: tetracycline transactivator; rtTA: reverse tetracycline transactivator.

Figure 3

Schematic of the rapamycin regulation system. The constitutively active human cytomegalovirus (hCMV) promoter drives expression of two fusion transcription factors. (Top left) A transcription factor consisting of three copies of the FKBP protein fused to a ZFHD1 DNA binding domain. (Top right) A transcription factor consisting of a FRAP protein with a p65 activation domain. Rapamycin enables dimerization of the transcription factors, with enable binding to 12xZFHD1 binding sites and activation, respectively, driving expression of a transgene upstream of a minimal CMV promoter. ZFHD1: zinc finger homeodomain-1; FKBP: FK-binding protein; FRAP: FKBP-rapamycin associated protein [23].

Figure 4

Schematic of the RU486 regulation system. A constitutively active promoter drives expression of a fusion protein consisting of a VP16 activation domain, a Gal4 DNA-binding domain (DBD) and a progesterone ligand-binding domain (Prog LBD). Binding of RU486 to the Prog LBD enables dimerization and binding to 4x Gal 4-binding sites upstream of a TATA box driving transgene expression [28]. Poly A, poly adenylation signal.