Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 243

North Carolina, USA, based biotechnology company. The
AAV-galanin program is currently in the late preclinical stage,
and no hard date has been set for clinical trial initiation. Other
approaches appear even further behind, but there is a feeling
that gene therapy is finally coming of age, and as the pharma-
ceutical industry embraces this technology, the clinical transla-
tion is likely to become far more rapid.

2 Materials

HEK293 cells 2.1 AAV Vector

Production

Plasmids

pFdelta6
pNLrepcap1
AAV pAM packaging plasmid

Complete DMEM/10% FBS

1 L Dulbecco's Modified Eagle Medium (DMEM, Life
Technologies).
10 ml sodium pyruvate.
10 ml nonessential amino acids.
100 ml certified heat-inactivated fetal bovine serum.
5 ml penicillin (10,000 U/ml)/streptomycin sulfate
(10,000 μg/ml).
Store at 4 °C in the dark for up to 2 months.
IMDM/5 % FBS

1 L Iscove's Modified Dulbecco's Medium (IMDM, Life
Technologies).
3.024 g sodium hydrogen carbonate.
50 ml certified heat-inactivated fetal bovine serum.
Store at 4 °C in the dark for up to 2 months.
2× HeBS buffer

11.9 g HEPES (50 mM final concentration).
16.4 g NaCl (28 M final concentration).
0.21 g Na 2 HPO 4 (1.5 mM final concentration).
800 ml tissue culture grade distilled water.
Titrate to pH 7.05 with NaOH.
Add water to 1 L.
Filter sterilize through a 0.22 μm nitrocellulose filter.
Aliquot 10 ml into sterile disposable polypropylene tubes and
store at −20 °C.