Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 114

4 Notes

1. Ammonium sulfate precipitation followed by CsCl gradients
was the traditional purifi cation method; however, CsCl gradi-
ent purifi cation is not scalable and takes 2 weeks to complete.
Additionally, CsCl is toxic to mucus membranes. Iodixanol is a
relatively expensive reagent but has a higher recovery than
CsCl. Heparin affi nity chromatography for AAV2 or AAV6
yields purer viral stock than that purifi ed with ammonium sul-
fate and CsCl, and additionally viral stocks has lower particle-
to-infectivity ratios.
2. Although some protocols allow freezing the pellet at this point,
we recommend not to freeze here and immediately move on to
the next step.
3. When purifying rAAV, rAAV aggregates with proteins in the
crude cell lysate. This has been found to change its buoyant
density and leads to distribution of the viral vectors along the
whole length of the gradient. To solve this problem, NaCl is
added to the fi rst iodixanol step (15 %) to destabilize ionic
interactions between macromolecules. NaCl is not added to
the other steps, such that the virus is banded under iso-osmotic
conditions and can be transferred directly in the following
steps ( see ref. 63 ).
4. A band of debris may form between the 40 and 20 % iodixanol
phases. Do not aspirate this band; aspirating may result in
blockage of the Sepharose column.
5. AAV2, AAV3, and AAV6 are known to bind to heparin, so
heparin column chromatography may be used for their purifi -
cation. If purifying other serotypes, e.g., AAV1, another type
of purifi cation must be used such as ion exchange chromatog-
raphy. One of the drawbacks of using heparin chromatography
is that other proteins with affi nity for heparin sulfate may co-
elute with AAV. This is especially relevant to methods which
use insect cells for AAV production, because heparin may bind
to baculovirus, which is used for infection of the insect cells
and must be subsequently purifi ed [ 28 , 109 ]. Additionally,
HPLC has previously been used by some researchers for AAV
purifi cation; however, the high pressure of HPLC is not suit-
able for viral particles.
6. Viral particle load should be adjusted according to your protocol;
higher viral particle loads will transduce cells more effi ciently;
however, higher viral load may lead to transduction of unintended
cell types or brain areas.