Biomedical Engineering Reference
In-Depth Information
primate striatum are suffi cient to protect DA neurons and attenu-
ate behavior defi cits [
23
].
In the present study, we have investigated whether the delivery
of AAV5 encoding GDNF would be feasible as a mean to intro-
duce suffi cient amounts of GDNF at the correct place.
2
Materials and Methods
2.1 Generation
of rAAV
For detailed description of vector preparation, please also see other
chapters in this volume. Briefl y, for preparation of the AAV vector
encoding for GDNF or GFP, the expression cassette was packaged
into AAV serotype 5 at uniQure facilities and produced with their
dedicated procedures. The expression cassette contains the cDNA
of the human GDNF gene, isoform 1, which is the longest isoform
of 636 bp, coding for the pre-proform. The expression is under the
control of the CAG promoter, a combination of the cytomegalovi-
rus (CMV) early enhancer element and chicken beta-actin pro-
moter. The GDNF is preceded by a Kozak sequence and followed
by the bovine growth hormone polyadenylation (BGHpA) signal.
The whole cassette is fl anked by two noncoding inverted terminal
repeats of AAV2. No additional regulatory elements were added
for ease in possible future regulatory approval for use in human. In
the control vector, the gene encoding the GDNF cDNA was sub-
stituted for the enhanced green fl uorescent protein (EGFP) cDNA.
Recombinant AAV5 vectors were prepared using a baculovirus
expression system, as described earlier [
50
-
52
]. Briefl y, three
recombinant baculoviruses, coding for REP, CAP5, and GDNF,
were used to infect SF9 insect cells. Purifi cation was performed using
AVB Sepharose high-performance affi nity medium (GE Healthcare,
Piscataway, NJ). The vectors were titrated using qPCR with primer-
probe combinations against the transgene and were expressed as
genomic copies per ml (GC/ml). Titers were in the range of
1-3 × 10
13
GC/ml for both vectors.
2.2 Animal Models
All experiments described were approved by the local authorities.
For testing of the AAV vectors, mice and rats are used. Rodents are
mostly used to obtain proof-of-concept and biodistribution data,
whereas the use of primates should be as restricted as possible. In
these studies, we have used untreated as well as 6-OHDA-treated
rats for proof of concept and demonstration of biological activity.
Two weeks after the AAV infusion, rats received a unilateral injec-
tion of 20
l of 6-OHDA (Sigma; calculated as freebase,
dissolved in ice-cold saline with 0.02 % ascorbic acid) into the right
striatum. The toxin is kept on ice, used fresh, and protected from
light to minimize oxidation. Care needs to be taken to avoid any
contact with the toxin itself.
ʼ
g/3
ʼ
Search WWH ::
Custom Search