Regulated Gene Therapy

Fig. 1

Schematic representation of the Tet-Off (left ) and Tet-On (right ) system, in the presence (higher panel ) or absence (lower panel ) of tetracycline (red squares). Tet-Off: the transactivator (tTA) is expressed under the control of a specific promoter. In the absence of tetracycline, it binds the operator (TetO) and activates the CMV promoter, leading to transgene expression, while in the presence of tetracycline, the tTA undergoes conformation changes and is no longer able to bind the TetO which ends the transcription of the transgene. Tet-On: in the absence of tetracycline, the reverse transactivator (rtTA) is unable to bind the TetO and no transcription can occur, when in the presence of tetracycline it changes conformation and is enabled to bind the TetO, allowing transcription of the transgene

Although both systems are commonly used in neuroscience research, it is considered preferable to use a Tet-On approach for the development of gene therapy for the treatment of neurological disorders. Indeed, an approach where transgene expression is normally repressed and will only occur when patients are submitted to treatment with the inducer is considered safer. The original inducing drug used to activate the Tet-On system was a tetracycline, but other derivatives have been used. Among theme, doxycycline, another antibiotic, is currently the most widely used as it has a low cost and a long half-life and crosses the blood-brain barrier easily [5]. However, it has been shown that the half-life of doxycycline can be reduced by 50 % when co-administered with other neurological treatments [6]. Patients suffering from neurological disorders are usually treated with various cocktails of drugs. It is therefore important to bear in mind that inducers remain active drugs, which could interact with other treatments that the patients might be on. Doxycycline has very limited side effect and has been safely used in the clinic. In rodents, it can be administered by gavage (20–50 mg/day) or through drinking water (200 μg to 2 mg/ml) [7–9]. Importantly, if the doxycycline is administered via drinking water, sucrose (2–5 %) should be added to cover the bitter taste of the drug and water bottles should be changed every other day as the drug loses stability over time. In the clinic, doxycycline is administered orally, with doses between 100 and 200 mg/day for adults. It is of course important that the dose of inducer required to reach therapeutic level of transgene expression remains below that threshold. Prolonged use of antibiotics as inducer rises important issues, not only in terms of side effect for the patients, but it also increases the risk of promoting the development of antibiotic resistance in bacteria strains [10]. For that reason, scientists have been looking at alternatives. The use of the doxycycline metabolite (4-epidoxycyclin) or the tetracycline agonist (GR333076X), which has no antibiotic properties, is a promising option.

1.2 The Five Golden Rules for a Clinically Relevant Inducible Transgene Expression System

Absence of basal expression: In order to be safe, the expression should be as close to zero as possible in absence of the inducing drug (“off” state). It is indeed crucial to ensure that the residual level of expression of transgene remains below therapeutic action so the system can be shut down in case of adverse effects.

Rapid, dose-dependent induction: The expression of the transgene should occur rapidly after administration of the inducing drug. The level of expression of the transgene should be dependent of the dose dependency inducer administered. The inducer should be have a long half-life and be able to cross the blood-brain barrier. Finally, protein levels should be within the therapeutical range.

Quick shut down: In order to better manage potential side effects, the expression of the transgene should stop rapidly after discontinuation of the drug treatment. However, the stability of the therapeutic molecule will also influence the duration of ongoing adverse events.

Specificity: The transgene should be expressed in a discrete area and/or cells types in the brain . This will ensure maximal and localized effect while reducing the risk of adverse effect.

Limited immune response: The delivery of viral vectors into the brain requires surgical intervention, thus compromising the blood-brain barrier. The rupture of the wall, normally isolating the brain from circulating white blood cells, might trigger an immune response against the exogenous protein. To minimize immunogenicity, it is important to use human genes and to avoid contamination with animal products (e.g., serum in culture medium).

1.3 In a Nutshell

Main advantages: The Tet system allows fine control of level of expression of the transgene and can be shut down if necessary. Doxycycline is commonly used in the clinic to treat infections; it is a potent, low-cost, cheap, and safe inducer. Finally, the Tet system has been extensively characterized and tested in animal model s of neurological disorder (for review, see ref. 11).

Main drawbacks: One of the main challenges associated with inducible expression approaches for gene therapy is the leakiness of the system. The existence of basal level of expression of the transgene in “off state” raises serious concern about the controllability of the system. Research is currently ongoing to improve tightness of the system, including reduction of nonspecific transactivator-TetO binding. The second issue concerns the triggering of an immune response and inflammation by the Tet transactivator. As the majority of the population have been in contact with the herpes simplex virus from which the VP16 part of the rTA has been derived, this system can be particularly immunogenic [12].

Important things to consider: The Tet systems comprise two elements, the rtTA and the transgene cassettes (Fig. 1) that can be delivered either separately or by the same vector. However, it is possible that altogether, these constructs exceed the cloning capacity of certain viral vectors (e.g. adeno associated virus ≈4.5 kb). Although the use of a dual-vector approach is possible, it results in reduced expression of the transgene, as each cell has to be transduced by the two vectors to allow gene expression. The single vector approach is therefore highly recommended. Different configurations of the Tet system have been developed and adapted for single vector approaches: either using right-facing cistrons, where the transgene and the rtTA are placed one after the other, or using a bidirectional promoter to drive the expression of the transgene, on one side, and the rtTA on the other side. Finally, the areas of the brain and the cell types to be targeted should be carefully chosen as ectopic expression can influence efficacy and safety of the treatment.

1.4 Autoregulated Promoters

An autoregulated system could be an alternative to using a drug-regulated system, such as the tetracycline and rapamycin systems. These have the advantage that no proteins of nonmammalian origin have to be over expressed and no exogenous administration of a regulating drug is required. Instead, autoregulated systems are based on a promoter or regulatory elements from an endogenous gene. Examples of promoters that have been used in autoregulated vectors for CNS gene therapy are the glial fibrillary acidic protein (GFAP) promoter and the enkephalin (ENK) promoter [13]. GFAP is expressed in astrocytes and is upregulated in the gliotic reaction following a lesion. ENK has been shown to be upregulated in striatal neurons of the indirect pathway following dopamine depletion in Parkinson’s disease (PD). The authors showed that vectors containing these promoters have a similar expression pattern in rat striatum as the endogenous proteins in animals subjected to lesions or dopamine depletion, respectively. The transgene expression was also responsive to decreases in inflammation and restoration of dopamine levels. The hypoxia-responsive element (HRE) from the erythropoietin gene has also been used to achieve autoregulated transgene expression in the CNS [14]. In this system, nine copies of the HRE sequence were coupled to a SV40 minimal promoter. No transgene expression was detected in healthy mouse brain , but transgene expression could be detected following transient middle cerebral artery occlusion. These results show that promoter elements or promoters of genes regulated by disease or changes in the cells environment could be used to create autoregulated vectors.

Zinc finger-based transcription factors (ZFTFs) can be used to regulate the transcription of endogenous genes. ZFTF consists of several connected zinc fingers, which determine binding specificity, a nuclear localisation signal and an activating or repressing domain. A ZFTF consisting of six zinc fingers recognizes an 18-base pair sequence and is regarded as specific for one site in the human genome. Most studies using ZFTF in the CNS have used ZFTF designed to target and upregulate the endogenous vascular endothelial factor (VEGF ) gene. VEGF plays a role in angiogenesis, but has also been shown to have neuroprotective and neurotrophic effects [15]. Beneficial effect on cell survival and motor behavior has so far been reported in rat models of stroke, spinal cord injury, and traumatic brain injury following injection of viral vectors carrying the VEGF ZFTF gene [16–18]. ZFTF has also been used in studies on the neurodegenerative disorders Huntington’s disease (HD) and PD. In the HD study, the authors used a ZFTF designed to target extended CAG repeats [19]. By this approach, they were able to specifically knockdown the mutant huntingtin allele and improve motor behavior in an HD mouse model. In the PD study, the authors used a ZFTF designed to upregulate the endogenous glial cell line-derived neurotrophic factor (GDNF ) gene [20]. This potent neurotrophic factor has been shown to promote the survival of dopaminergic neurons. Expression of the GDNF ZFTF in a rat model of PD reduced the loss of dopaminergic neurons and improved motor behavior.

Transcription activator-like effector based transcription factors (TALE-TFs) can also be used to regulate the transcription of an endogenous gene. TALE-TFs consist of several connected DNA-binding repeats derived from natural TALEs found in Xanthomonas, a nuclear localization signal and an activating or repressing domain. The DNA-binding domain of TALE-TFs is more modular than the domain found in ZFTF. While each finger in a ZFTF recognize three to four base pairs and neighboring fingers affect each other, each DNA-binding repeat in a TALE-TF recognise only one base pair without any influence from neighboring repeats. The use of TALE-TFs to regulate endogenous genes is still a fairly new technology and studies using TALEs in the CNS has therefore been few. However, a recent study combined light-inducible transcriptional effector technology with a customised TALE DNA-binding domain to create an optically controlled TALE-TF [21]. The authors used a two-component system where the first component contained the TALE DNA-binding domain coupled to CIB1 and the second component contained a light-sensitive cryptochrome 2 protein coupled to an activator. Upon exposure to blue light, CIB1 and cryotochrome 2 combine to create a functional TALE-TF. The study showed that this technology could be used to upregulate the endogenous metabotropic glutamate receptor 2 gene (Grm2) in the mouse prefrontal cortex. Upregulation of Grm2 was also possible in a more traditional TALE-TF setting using a TALE DNA-binding domain coupled directly to the activator.

Both ZFTF and TALE-TFs have the advantage that all splice variants of the gene is produced since both technologies function at the level of transcription. This is essential when overexpressing certain genes. For instance, overexpression of VEGF using cDNA for only one splice variant leads to the formation of leaky vessels. By contrast, overexpression of all the splice variants using a ZFTF designed to target the endogenous gene leads to new fully functional vessels [22].

Only gold members can continue reading. Log In or Register to continue