Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 223

Fig. 7 Transduction of human retinal explants ex vivo by adeno-associated virus (AAV) serotypes. Images
showing retina transduced with rAAV2/5 expressing GFP at 11 days post-transduction. GCL ganglion cell layer,
INL inner nuclear layer, ONL outer nuclear layer. Scale bar 80 ʼ m

be imaged daily on an inverted microscope to assess transduction
and transgene expression if a vector containing a fl uorescent marker
such as GFP is used (Fig. 7 ). We have found that explants can be
maintained in a healthy state for at least 14 days.

6 Testing AAV Vectors In Vivo

The cellular tropism, kinetics, and transduction effi ciency of vec-
tors in the retina can only be defi nitively tested using in vivo mod-
els. There are numerous mouse models of retinal disease, a review
of which is outside the scope of this chapter. However, tropism and
effi ciency of vectors may differ according to the species [ 38 ] and
model used; therefore, using a model that most closely represents
the disease investigated would give the most reliable indication of
vector effi ciency. Below, we describe techniques for delivering vec-
tor to the murine eye and assessing transduction using in vivo con-
focal scanning laser ophthalmoscopy and a suggested protocol for
processing histological sections.

There are several different techniques of intraocular injection that
may be used in the delivery of AAV to the eye. Injections may be
directed into the vitreous or the subretinal space depending on the
target cell for gene therapy. The following is that which is used in
our laboratory.
Intraocular injections are performed following general anes-
thesia administered via an intraperitoneal route, under direct visual
control using a surgical microscope. A circular cover glass (Ø6mm,
VWR International) is applied onto the cornea with a carbomer
coupling gel (Viscotears, Novartis) to ensure a good visualization
of the fundus. The eye position can be controlled and stabilized by

6.1 Intraocular
Injections