Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 216

throughout the entire CNS [ 43 ], including the spinal cord, using
vector doses that are lower than those administered via the blood
circulation.
In some instances, it is required to limit transgene expression
to the cells of interest. Specifi c routes of delivery can restrict expres-
sion of the transgene to subsets of neurons. For instance, motor
neurons innervating a particular muscle group can be selectively
targeted using retrograde infection from the injected muscles.
However, in most cases it is needed to apply additional strategies
to limit transgene expression to a certain cell population. Capsid
composition determines vector affi nity for target cells. Hence, the
selection of the AAV serotype, among >100 different serotypes
described nowadays, will critically determine both the diffusion
and tropism of vector particles.
The expression pattern can be further modifi ed using pro-
moter systems for cell-specifi c transcriptional activity [ 44 , 45 ].
Promoters with a minimal size, such as the synapsin promoter, can
drive transgene expression in neuronal cells only and be adapted to
small size viral particles including AAV vectors [ 46 ]. Addition of
sequences in the transcribed portion of the transgene to destabilize
mRNAs by miRNA targeting (“detargeting” system) further
restricts expression to particular cell types [ 47 ].
Viral vectors for effective gene delivery to motor neurons will
be crucial tools to investigate the cause of MNDs and design thera-
peutic strategies. In order to transduce motor neurons broadly dis-
tributed along the motor pathway, it is crucial to carefully explore
combinations of vector type and route of delivery to optimize spe-
cifi c targeting of spinal and cortical motor neurons. AAV vectors
represent the most advanced vector type for clinical applications of
gene therapy against CNS disorders. Here, we will focus on the
description of AAV serotype 6 (AAV6) vector production and
injection for infection of motor neurons. Indeed, AAV6 vectors
can effectively transduce neuronal cells and achieve long-term
transgene expression. In particular, these small-sized particles are
effi ciently retrogradely taken up by axons and synaptic terminals
following intramuscular and ICV injections.

2 Materials

1. Cells . HEK 293-AAV cells (Agilent Technologies, Santa Clara,
CA, USA; #240073)
2. Plasmids . pAAV-cmv-GFP (Agilent) ( see Note 1 ); pDP6
(derived from pDP6rs, PlasmidFactory GmbH & Co.,
Bielefeld, Germany; #PF406)
3. Petri dishes . 500 cm 2 Petri dishes (Dishes Nunclon™

2.1 AAV6 Vector
Production

Δ , NUNC

A/S, Roskilde, Denmark; #166508)