Functional Neuroprotection and Efficient Regulation of GDNF Using Destabilizing Domains in a Rodent Model of Parkinson’s Disease

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has great potential to treat Parkinson's disease (PD). However, constitutive expression of GDNF can over time lead to side effects. Therefore, it would be useful to regulate GDNF expression. Recently, a new gene inducible system using destabilizing domains (DD) from E. coli dihydrofolate reductase (DHFR) has been developed and characterized. The advantage of this novel DD is that it is regulated by trimethoprim (TMP), a well-characterized drug that crosses the blood-brain barrier and can therefore be used to regulate gene expression in the brain. We have adapted this system to regulate expression of GDNF. A C-terminal fusion of GDNF and a DD with an additional furin cleavage site was able to be efficiently regulated in vitro, properly processed and was able to bind to canonical GDNF receptors, inducing a signaling cascade response in target cells. In vivo characterization of the protein showed that it could be efficiently induced by TMP and it was only functional when gene expression was turned on. Further characterization in a rodent model of PD showed that the regulated GDNF protected neurons, improved motor behavior of animals and was efficiently regulated in a pathological setting.

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“…The TMP-protein regulation approach does not suffer from these weaknesses. Thus far, studies using TMP regulation in the rodent brain have shown that there are no immunogenic properties associated with the destabilization domain (32). Furthermore, this system has the ability to be finely tuned for tight control of transgene expression by adjusting TMP dosing and expression can be reversed if needed, addressing a major barrier in the development of neuroprotective gene therapies.…”

“…Combining this approach with a high-expressing promoter, we can reduce the potential silencing effects seen in low-expressing promoters. This makes the system attractive for use in the central nervous system (32,33).…”

“…Recently, drug-regulated destabilization domains have been shown to control transgene expression in the brain (31)(32)(33). This system requires fusing the E. coli dihydrofolate reductase (DHFR) gene to the transgene of interest to produce a fusion protein.…”

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“…Moreover, with the use of DD-ecDHFR/TMP, Quintino and collaborators were able to control the level of glial cells-derived neurotrophic factor (GDNF), a protein that exerts neurotrophic and neuroprotective effects on dopamine neurons, in a rodent model of Parkinson's disease (Quintino et al, 2013) confirming the great therapeutic potential of GDNF against Parkinson's disease.…”

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“…In all these studies, the inducer doxycycline was applied for many days or even weeks over the drinking water, it is thus unknown if shortterm doxycycline applications would result in similar effects. In an alternative system depending on stabilization of GDNF fusion proteins coupled to destabilizing domains of E.coli dihydrofolate reductase (DHFR), expressed from a LV vector, striatal GDNF levels of about 125 pg/mg tissue were achieved in the trimethoprim-activated ON-state, while background was low at about 13 pg/mg tissue 33 . In yet another approach, a rapamycinregulated AAV vector expressed GDNF up to 450 pg/mg tissue, without any non-induced background, although by exploiting clinically not acceptable dosages of rapamycin 7 .…”