TRANSCIPTIONAL REGULATION OF TRANSGENE EXPRESSION

The promoter, defined as a fixed region located no more than 100 bases from the transcription start site, contains three basic elements. These elements include a TATA box, a CAAT box and a GC box. The TATA box is located 10-30 bases upsteam of the transcription start site. It functions unidirectionally with ATATAA as its consensus sequence. The CAAT box, located about 80 bases from the transcription start site, can function in either orientation with GGCCAATTC as a typical consensus sequence. Multiple GC boxescan be found throught the promoater region but are generally located 100 bases from the transcription start site. The GC box consensus sequence is GGGCGG and can function in either orientation.

Enhancersare bidirectional elements located at multiple sites within a gene. It is not uncommon to find these elements within an intron or the 3' untranslated region of a gene. Other regulatory elements tend to be found only upstream of the transcriptional start site. Both response elements and upstream activation sequences are typically located within 1 kilobase of the transcription start site and can confer either tissue specific expression or coordinate expression between multiple genesLocus control regions, which are generally far removed from the transcriptional start site of a particular gene, can also play arole in coordinating expression between multiple genes.

All of these regions can be modified to regulate transgene expression. Our knowledge of these regions is still evolving and unique combinations may prove beneficial in regulating transgene expression in a particular cell population. Currently, either tissue specific or tumor specific promoaters have been used to regulate gene expression within a desired cell type. Unfortunatly, these promoater are much less efficient at transgene expression than constitutive promoaters such as CMV. Also, as stated during the lectures on transgenic animals, the endogenousgenetic enviroment surrounding the transgene can greatly influnce its expression such that even robust promoaters such as CMV may not yield high levels of gene expression under all circumstances.

Combining tissue-specific or gene family specific upstream activation sequences with constitutive promoaters can limit robust gene expression to a particular cell type. Myogenic activators, such as those of muscle creatine kinase have been combined with the CMV promoater to result in high levels of gene expression only in muscle.

Unfortunatly, none of these systems has the ability to be temporally regulated. Particular gene therapy applications might require regulated gene expression. Currently two systems have been described which enable some control over turning on or turning off transgene expression. These systems induce gen expression by using either the steriodal hormone or tetracycline responsive systems. Other inducible systems such as isopropyl ß-D-thiogalactoside, or heavy metals have either the potential to activate endogenous genes, or are too toxic or inefficient for in vivoapplications. These systems have become known as 'gene switches' and the ultimate switch for gene therapy should meet the following criteria:

• Transgene activated only upon administration or removal of an exogenous compound.
• The exogenous compound should be non-toxic and preferably orally administered.
• The exogenous compound only activates the transgene and does not interfere with endogenous gene expression.

Given these criteria we will explore the two reported gene switches, the progesterone-responsive switch and the tetracycline-responsive switch.

The Progesterone Responsive Switch.

Exogenous compound= RU486m

Basics- A mutant progesterone receptor (hPRB89) binds RU486 but not progesterone yet activates transcription in response to ligand binding. RU486 levels required for active transcription are much lower than that required for anti- progesterone or anti- glucocorticoid activity

Components - A two plasmid system. The chimeric GL or GL-VP protiens, produced by the regulator constructs, binds to upstream elements contained on reporter constructs. The GL or GL-VP binding initiates transgene expression from the reporter construct.

Regulator Constructs - Produce either GL or GL-VP protiens, expression driven by CMV promoter

Reporter Constructs

Findings - Both TATA and tk promoters had basal expression in vitro which was increased 10 fold after RU486 addition. Maximal transgene expression levels were observed for the tk constructs. Expression was turned on 2 hours after RU486 addition and reached maximal levels at 8 hours. Down regulation of transgene expression was not mentioned in the initial studies but more recent work suggests that expression remains on for at least 24 hours after the last dose of RU486. Studies were done both in vitro and ex vivo

The Tetracycline Responsive Switch

Exogenous compound= tetracycline

Basics - Tetracycline repressor binds to the tetracycline-resistnace operator in absence of tetracycline to initiate transcription. Addition of tetracycline turns off expression.

Components - Two plasmid system. Regulator constructs produce the tTA chimeric protien which binds to tetracycline operator found on reporter constructs.

Regulator constructs

Reporter constructs