Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 219

This is the “wash” tube and may have a signifi cant amount of AAV
remaining in it.
An SDS-PAGE analysis of the isolated virus and wash sample
can be conducted as an indicator of purity. Dilute the samples 1 in
10 and heat with Laemelli buffer at 95 °C for 10 min. Run on a
10 % Tris-HCl gel at 100 V for 2 h and then stain with EZBlue
(Sigma). This should reveal three bands representing the viral cap-
sids, VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa) (Fig. 5c ).

The sample of virus should be prepared by digesting with a DNase
enzyme to remove any unpackaged plasmid DNA: add 1

4.7 Virus Titer
Using qPCR ʼ l sample

of undiluted virus to 1 ʼ l DNase enzyme (New England Biolabs,

NEB), 1 l H 2 O and then incubate at

37 °C for 1 h. Subsequently dilute the sample 1:10 with H 2 O, and
incubate at 95 °C for 15 min to denature the sample.
Many real-time PCR machines contain software allowing for
quick and easy viral titer determination, but this can also be done by
exporting generated Ct data to Excel. Design primers to bind to a
region of the transgene, and conduct a dilution series of a positive
template control (10 ng, 1 ng, 100 pg, and 10 pg should be suffi -
cient). Using the denatured virus sample (which is now at a dilution
of 1:100), set up triplicate reactions per sample, adding 1

ʼ l buffer (NEB), and 7 ʼ

l dena-
tured virus to each reaction. Use standard SYBR Green methods,
and when the reaction is complete, select a consistent threshold
incorporating the log phase of each reaction; this will select the com-
parative Ct value for each sample at that threshold. To determine
titer without using a supplied software, generate a standard curve for
the positive template control. To do this, convert the positive tem-
plate control from nanograms into copies of amplicon: divide mass
of template (g) by the molecular weight of amplicon (g/mol), and
multiply by Avogadro's constant to generate the number of mole-
cules. Convert into log values and plot against the Ct values from
the dilution series to generate the standard curve. Ct values of the
diluted virus samples can then be compared to the standard curve
and equivalent molecules determined. Convert the log value back to
the actual number of molecules and account for the dilution factor.
Multiply by 1,000 to give genome particles per ml.

ʼ

4.8 Site-Directed
Mutagenesis to Form
Mutant Capsid
Variants

To create capsid mutant vectors, site-directed mutagenesis of the
plasmid containing the AAV capsid gene may be performed using
a commercial kit such as the QuikChange II XL Site-Directed
Mutagenesis Kit (Agilent Technologies), which is suitable for large
plasmids. The steps involved are detailed below and based on the
manufacturer's guidelines.

Two oligonucleotide primers should be designed so that they both
contain the desired capsid mutation and anneal to the same
sequence on complementary strands of the plasmid. Primer
sequences for rAAV2/2 single capsid mutation are available in the

4.8.1 Primer Design