AAV Gene Therapy Strategies for Lysosomal Storage Disorders with Central Nervous System Involvement - Gene Delivery and Therapy for Neurological Disorders - page 275

(k) Note level of virus in syringe after injection. Ensure that
appropriate virus volume was delivered.
(l) Alternatively, the incision can be closed with sutures.
(m) Once the mouse has awoken from anesthesia and is active,
place it in normal cage setup with gel pack and food.
(n) Alternatively, remove sutures if used.

2.2 Injection of AAV
Vectors into
the Cerebral Lateral
Ventricles of Neonatal
Mice

Infusion of AAV vectors into the cerebral lateral ventricles (intracere-
broventricular route) of neonatal mice is an outstanding approach
to achieve widespread gene delivery throughout the brain [ 85 ]. The
advantage of intracerebroventricular delivery in neonatal mice for
LSDs is the diffuse nature of CNS gene transfer [ 66 , 85 , 86 ] and
early intervention at an age that may precede the onset of disease
symptoms. In addition, the degree of neuronal gene transfer achieved
by ICV delivery is considerably higher than achieved by systemic
administration of the new blood-brain barrier-crossing AAV vectors
(AAV9, AAVrh10, and others) [ 67 , 73 ] using total doses 10-100-fold
lower. CSF infusion of AAV vectors in adult mice shows limited dis-
tribution in the CNS [ 87 ], but recent studies in pigs and monkeys
have shown that intrathecal or cisterna magna infusion of AAV9 vec-
tors may be an effective route to achieve widespread transduction in
the CNS [ 78 , 88 ]. The therapeutic effectiveness of neonatal ICV
infusions has been demonstrated in a number of mouse models
of LSDs, including MPS VII [ 85 ], Krabbe disease [ 89 ], GM1-
gangliosidosis [ 90 ], and mucopolysaccharidosis type I (MPS I) [ 91 ].
This CNS delivery route, especially in the context of AAV-encoded
enzymes for the treatment of LSD, is both potent and viable.

Sterile AAV vector to be injected
10

2.2.1 Material
and Reagents

L NanoFil syringe (World Precision Instruments)*
36 gauge beveled needle (NF36BV-2; World Precision
Instruments)*
Kimwipes
70 % ethanol
0.5 M sodium hydroxide
Isofl urane
Fiber-optic illuminator (optional)
*Autoclave before use

ʼ

1. Remove the mother from the cage with the pups, and put in a
separate clean cage.
2. Place two pieces of Kimwipes into the bottom of a 50 mL con-
ical tube. Add 1 mL of isofl urane onto the Kimwipes, and then
add another piece of tissue.

2.2.2 Protocol