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Figure 6 Formation of vascular structures in VEGF-myoblast-implanted legs. Myoblasts expressing the murine VEGF164 gene were injected into mouse hindlimb. Histological analysis of injected muscles were conducted at day 44-47 postimplantation using hematoxylin/eosin staining of cryostat sections. Uninjected control legs were normal both in size and in morphology (left panel), whereas legs injected with VEGF myoblasts (right panel) were greater than twice the diameter of control legs, and consisted primarily of hemangioma and pools of blood. Both panels are shown at the same magnification. These results demonstrate the importance of regulating recombinant gene expression in gene therapy applications. (Adapted and reprinted from Ref. 110 with permission, copyright 1998 Cell Press.)

In its original and simplest form, the tet transactivator (tTA) is a hybrid factor comprising a bacterial tetracycline repressor (tetR) and the viral transactivator domain VP16 (123). When bound to tet, tTA is prevented from binding to tet operator sequences juxtaposed to a minimal promoter, and gene expression is turned off. In the absence of tet, tTA is free to bind to the inducible promoter, and gene expression is induced up to 5 or 6 orders of magnitude. A relatively recent modification of the system allows for induction of gene expression in the presence, rather than absence, of tet (124). A second chimeric protein containing a mutated version of tetR was developed and designated as ''reverse'' tTA (rtTA); this transactivator binds to tet operator sequences in the presence of tet. Both tTA and rtTA have been demonstrated to efficiently regulate expression in tissue culture, fruit flies, and mice (125-128).

A major advance in broadening the utility of the tet system was the employment of retroviruses. Retroviral gene delivery is much more rapid and efficient than transfection-using plasmids. Retroviral vectors also do not form concatemers and thus should not form a repressive chromatin environment sometimes associated with plasmids (129). Genes can be introduced into tens of thousands of myoblasts at high efficiency, generating polyclonal populations within a week (50), an advantage over the few stable clones routinely obtained. For these reasons, retroviruses are well suited for delivery of tet-inducible systems to primary cells isolated directly from tissue. Initial studies using tet-regulatable cassettes, however, met with numerous problems. In one case, the inclusion of an autoregulatory feedback loop necessitated high background levels of expression in order to ''jumpstart'' the system (130). In other cases, overcomplexity of transcription and translation units produced low viral titers (131-135).

Bohl and colleagues (136) first overcame this problem by using simplified retroviral vectors in which the necessary elements were dispersed over more than 1 retroviral vector. In this study, 1 retrovirus encoded rtTA, whereas the other contained an inducible Epo cassette. After multiple rounds of infection, primary myoblasts exhibited induction of about 200-fold in expression of the protein. When the engineered myoblasts were transplanted into mice, Epo expression could be repetitively turned on and off over a 5-month period by controlling levels of dox in drinking water. In an improvement of this approach, inclusion of a selectable marker such as green fluorescent protein (GFP) allows for purification by flow cy-tometry of regulatable populations of cells (128) (Fig. 7).

Recently, 2 additional advancements of the tet system have increased its applicability for gene therapy purposes. The tetR transcriptional elements are modular; one may replace the VP16 transactivator domain of tTA, for instance, with a KRAB transrepressor domain to create a tet-regulated repressor of transcription (137). Expression of 2 tet modulators within the same cell, however, leads to formation of nonfunctional heterodimers because the modulators have identical di-merization domains (Fig. 8) (138). Based on sequence information and known crystal structures of tetR as well as mutational analysis (139-141), mutually distinct dimerization domains deriving from separate classes of Gram negative bacteria have been identified (142,143). The ability to engineer tet modulators with specific dimerization domains allows tet activators and tet repressors to be expressed within the same cell without risk of forming a nonfunctional heterodimer. The development of such a tetracycline-inducible retroviral system, designated the RetroTet-ART (activators and repressors expressed iogether) system (142), allows for gene expression to be completely extinguished or induced in a fully dose-dependent manner—as a result, the dynamic range of gene expression has been greatly increased (Fig. 9). This improvement is a significant advantage in applications where basal expression from the inducible promoter must be extinct. The RetroTet-ART system was demonstrated to be able to reversi-bly silence expression of p16, a growth arrest protein (142).

In a second modification of the tet-inducible system, the DNA-binding domain of tetR was altered to interact with a modified tet operator sequence (143). The original and adapted binding sequences were engineered into tTA and rtTA proteins harboring distinct dimerization domains. By placing 2 separate genes under control of old and new tet operator sequences and expressing both of the modified tTA and rtTA proteins, Baron and colleagues were able to either repress expression of both genes or express either gene alone simply by changing the dox concentration (Fig. 10) (143). A means of turning on both genes at once has yet to be achieved. Thus, the activity of two different genes can be reversibly controlled in a mutually exclusive manner.

In summary, tet-regulatable retroviral systems are capable of being repressed and expressed in a fully inducible manner both in vitro and in vivo. Tet has been used for decades in humans and animals, and only at higher doses above those required for induction of transgenes have few if any deleterious effects been observed. In addition, when the rtTA protein was delivered to mice by ex vivo gene delivery using my-oblasts, no immune response to foreign elements was observed (136). Retroviruses are efficient means of delivering tet-regu-latable vectors to large numbers of primary cultures of cells, including myoblasts. Thus, there is much reason to believe that the tet-regulatable retroviral systems, in conjunction with myoblast-mediated gene delivery, may be well suited for gene therapy applications in humans in the future.

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