Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 203

Fig. 9 Expression of mutant Htt in vivo. After the injection of LV-VSV-G encoding Htt171-82Q, aggregates can
be detected as soon as 1 week later and for up to 12 weeks. EM48 polyclonal antibody (not commercially
available) was used [ 33 ]

75 mg/kg-10 mg/kg and 150 mg/kg-10 mg/kg, respectively.
The top of the head is shaved and the animals are placed in a ste-
reotactic frame. The skin is opened to reveal the skull and the
bregma is localized. Striatum coordinates are determined on the
basis of a stereotaxic atlas [ 109 , 110 ]. With the aid of a binocular
microscope, holes are drilled at the following coordinates (1) for
rat striatum: tooth bar, −3.3 mm; anteroposterior, +1 mm from
bregma; lateral, ±3 mm; ventral, −4.5 mm and (2) for mouse stria-
tum: tooth bar, 0 mm; anteroposterior, +1 mm from bregma; lat-
eral, ±2 mm; ventral, −2.5 mm. Before stereotaxic injection,
concentrated lentiviral vector stocks are thawed and resuspended
by vortexing and repeated pipetting. LV concentration is adapted
according to the envelope used for pseudotyping and the species.
Two to four times more MOK-G- than VSV-G-pseudotyped LV
should be injected [ 37 ]. We currently use up to 1,000 ng of
LV-MOK-G and 600 ng of LV-VSV-G for rat striatum and up to
800 ng of LV-MOK-G and 200 ng of LV-VSV-G for mouse stria-
tum. A total injection volume of 2-8 μ l is acceptable for rat stria-

tum and of 2-3 l for mouse striatum. These volumes are adapted

as a function of the structure targeted. The injection is performed
with a 10-

μ

l syringe (34-gauge blunt-tip needle attached to a poly-
ethylene catheter), at a fl ow rate of 0.25

μ

l/
min for mice. When the injection is completed, cannulas are left in
place for 5 min to ensure effective diffusion of the LVs and to
prevent refl ux. The needles are then slowly removed and the skin is
sutured. Finally, the animals are kept in a recovery box maintained
at 37 °C.
A rodent model of chronic HD has been developed, based on
LVs encoding mutant Htt (Htt171-82Q as in the in vitro model).
Stereotaxic injections are carried out with the VSV-G-pseudotyped
LV/SIN-PGK-Htt171-82Q-WPRE, with a total of 800 ng of p24
antigen in two 4

μ l/min for rats and 0.2 μ

l aliquots delivered to the rat striatum [ 33 ]. In
this model, cell death is observed over a period of 6 months and
mHtt protein production induces the formation of Htt aggregates
as little as 2 weeks after viral injection (Fig. 9 ).

μ