AAV Gene Therapy Strategies for Lysosomal Storage Disorders with Central Nervous System Involvement - Gene Delivery and Therapy for Neurological Disorders - page 280

16. Make a craniotomy above each DCN using a 20 G hypodermic
needle inserted vertically (i.e., at an angle perpendicular to the
stereotaxic apparatus). Advance the needle through the skull
and cerebellar tentorium f .
17. Use a sterile 100 L Hamilton syringe fi tted with a non-coring

needle (22-25 G) to inject the AAV vector. Lower needle
through the skull and into the target area at a rate of 1 mm/s,
using the micromanipulator affi xed to the stereotaxic unit.
18. Inject a total of 24

ʼ

L/
min. Raise the needle by 0.15 cm between each bolus, so that
the fi nal needle position is 1.1 cm ventral to meninges.
19. Leave the needle in the DCN for 1 min after fi nal injection,
and then raise it halfway. After another minute, withdraw the
needle completely over 30 s e .

ʼ L in 10-14 ʼ L boluses at a rate of 2 ʼ

ICV Injection

Due to the narrow lateral ventricle of cats ( 2 mm wide), ultra-

sound guidance is used to confi rm correct placement of the injec-
tion needle. Since the thalamic injection needle transects the lateral
ventricle on its descent, it may disrupt ventricular volume and
architecture. Therefore, it is prudent to perform ICV injection
prior to treatment of the thalamus.

≤

20. Make a 2 cm incision in the skin over the bregmatic
fontanelle.
21. Attach the ultrasound probe and biopsy guide to a microma-
nipulator on the stereotaxic unit. This requires a custom posi-
tioning device that we devised for this purpose.
22. Visualize the left lateral ventricle using an 8-5 MHz ultra-
sound probe.
23. Insert a 20 G, 2.5 spinal needle through the biopsy guide to

penetrate the skull and serve as an anchor and guide for the
injection needle.
24. Load AAV vector into a sterile 250

″

ʼ L Hamilton syringe fi tted

needle, and manually insert it through the
guide needle into the lateral ventricle. The needle bevel should
face dorsally for easier ultrasound confi rmation of proper place-
ment within the lateral ventricle.
25. Inject a total of 200

with a 25 G, 3.5 ″

L of AAV vector (with or without 4 %
gadoteridol in the injectate g ) in 10-15

ʼ

ʼ L boluses at a rate of

L/s, with ~1 min gap between boluses.
26. Leave the injection needle in place for 2 min after the fi nal
injection and then withdraw completely. Finally, remove the
guide needle e .

3-5 ʼ