Viral-Mediated Temporally Controlled Dopamine Production in a Rat Model of Parkinson Disease

Abstract

Regulation of gene expression is necessary to avoid possible adverse effects of gene therapy due to excess synthesis of transgene products. To reduce transgene expression, we developed a viral vector-mediated somatic regulation system using inducible Cre recombinase. A recombinant adeno-associated virus (AAV) vector expressing Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor alpha (CreER(T2)) was delivered along with AAV vectors expressing dopamine-synthesizing enzymes to rats of a Parkinson disease model. Treatment with 4-hydroxytamoxifen, a synthetic estrogen receptor modulator, activated Cre recombinase within the transduced neurons and induced selective excision of the tyrosine hydroxylase (TH) coding sequence flanked by loxP sites, leading to a reduction in transgene-mediated dopamine synthesis. Using this strategy, aromatic L-amino acid decarboxylase (AADC) activity was retained so that l-3,4-dihydroxyphenylalanine (L-dopa), a substrate for AADC, could be converted to dopamine in the striatum and the therapeutic effects of L-dopa preserved, even after reduction of TH expression in the case of dopamine overproduction. Our data demonstrate that viral vector-mediated inducible Cre recombinase can serve as an in vivo molecular switch, allowing spatial and temporal control of transgene expression, thereby potentially increasing the safety of gene therapy.

Efficacy of Codelivery of Dual AAV2/5 Vectors in the Murine Retina and Hippocampus

“…For example, in models of Parkinson's disease coexpression of three dopaminesynthesizing enzymes proved beneficial (Muramatsu et al, 2002). Furthermore, spatial and temporal control of transgene expression could be mediated by a second AAV vector expressing, for example, inducible Cre recombinase (Li et al, 2006). In the case of therapies for mendelian diseases in which the underlying mutation is unknown, a combination therapy of antioxidants and/or growth factors may prolong cell survival and function (Komeima et al, 2007;Yang et al, 2009;Li et al, 2010).…”

Compensatory Regulation of Dopamine after Ablation of the Tyrosine Hydroxylase Gene in the Nigrostriatal Projection

“…AAV Vector Construction-We generated two types of AAVCre vectors basically as described previously (19). One was the AAV-Cre vector, which contained an expression cassette with a human cytomegalovirus immediate early promoter (CMV promoter), followed by the first intron of human growth hormone, Cre recombinase cDNA, and simian virus 40 polyadenylation signal sequence (SV40 poly(A)), between the inverted terminal repeats of the AAV-2 genome.…”

“…A microinjection of adeno-associated viral (AAV) vector expressing Cre recombinase (AAV-Cre) (19,20) into the substantia nigra pars compacta (SNc) of the floxed Th mice disrupted the expression of the Th gene in a subset of neurons in the SNc of the adult mice. Our biochemical and histochemical analyses suggest two regulatory mechanisms of axonal TH for dopamine homeostasis in the nigrostriatal projection.…”

Controlled and localized genetic manipulation in the brain

“…The rAAV transduction vector strategy offers hope for some of these retinal diseases. In a rat model of Parkinson Disease, a combination of rAAV vectors has been used to temporally control dopamine production [81]. One vector expressed the regulated CreERT2 and the other carried a floxed gene for a dopamine synthetic enzyme, allowing selective KO of the synthetic enzyme, tyrosine hydroxylase, upon treatment with tamoxifen.…”