Herpes Simplex Virus 1 (HSV-1)-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 68

(FA), the most common recessive form of ataxia in humans, which
originates from a defi ciency in frataxin, a protein encoded by the
FRDA gene. A fi rst study [ 119 ] demonstrated that the synthesis of
frataxin could be eliminated in neurons from transgenic mice car-
rying “fl oxed” FRDA ( loxP - frda ) genes by infection with amplicon
vectors expressing CRE recombinase (CRE-amplicons). In vivo
delivery was achieved by stereotaxic injection of the CRE-amplicons
into the brainstem of loxP-frda mice, to generate a localized gene-
knockout model. These mice developed a behavioral defi cit detect-
able after 4 weeks, and when reinjected with amplicons expressing
the frataxin cDNA, they exhibit behavioral recovery as early as 4
weeks after the second injection. In a second study, amplicons were
used to deliver a 135-kb insert containing the entire 80-kb FRDA
human genomic locus, including long upstream and downstream
regulatory sequences (the FXN genomic DNA locus) into FA
patient defi cient primary fi broblasts [ 120 ]. Synthesis of frataxin in
the FRDA-transduced FA-deficient cells was confi rmed by
immune-fluorescence. Moreover, functional complementation
studies demonstrated restoration of the wild-type cellular pheno-
type in the FRDA-transduced cells in response to oxidative stress.
More recently, and to investigate the persistence of transgene
expression in the brain provided by the amplicon-delivered 135-kb
FXN genomic DNA locus, the same group constructed a second
vector carrying the 135-kb FXN locus but with the E. coli lacZ
gene inserted at the ATG start codon [ 121 ]. Direct intracranial
injection of this vector into the adult mouse cerebellum resulted in
a large number of cells expressing lacZ driven by the FXN locus,
which persisted for at least 75 days. In contrast, synthesis of GFP
expressed from the same vector, but driven by the HSV-1IE4/5
promoter, was strong but transient. This study demonstrated for
the fi rst time a sustained transgene expression in vivo by amplicon-
borne delivery of a very long genomic DNA locus.
Ataxia-Telangiectasia (AT) is an autosomal recessive disease
with a pleiotropic phenotype, characterized by cerebellar degen-
eration, immunodeficiency, cancer predisposition, radiation
sensitivity, and premature aging. This disease is caused by a defect
in the ATM (Ataxia Telangiectasia Mutated) gene, which is respon-
sible for recognizing and correcting errors in duplicating DNA
when cells divide. Currently, no treatment can stop progression of
AT. Expression of the ATM cDNA from amplicons allows func-
tional recovery of human AT fi broblasts [ 122 ]. In a further study
from this team, an amplicon encoding both the enhanced green
fl uorescent protein (EGFP) and a human FLAG-tagged-ATM pro-
tein was inoculated in the cerebellum of Atm −/− mice. This ampli-
con was delivered to thousands of cerebellum cells, including
Purkinje cells, as assessed by EGFP fl uorescence. FLAG-tagged-
ATM expression was demonstrated at transcriptional (qRT-PCR,
in situ hybridization) and translational (immune-precipitation of