Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 211

containing these larger genomes is enhanced by a treatment with
proteasome inhibitors. This effect was also observed in mouse and
dog models of hemophilia when treated with AAV vectors contain-
ing oversized genomes of 5.6 kb [ 54 ].
In 2008, Allocca et al. offered hope that AAV could package
genomes of up to 8.9 kb [ 55 ]. Their data initially suggested that
an Abca4 transgene was effi ciently packaged within different AAV
serotypes and effectively transduced Abca4 −/− mice. 8.9 kb trans-
gene fragments were detected by a Southern blot analysis and full-
length Abca4 isolated from injected Abca4 −/− retina. Despite these
data, later studies revealed it was highly unlikely that AAV was
packaging the full-length 8.9 kb transgene. The data observed
were found to be due to a recombination of partially packaged
transgenes within the host cells [ 56 - 58 ]. Strand-specifi c probes
used to detect single-stranded AAV genomes revealed packaging
that occurs primarily from the 3 end until the capsid contains its

maximal capacity of ssDNA [ 57 ]. Despite this fi nding, the above
studies all detected the full-length expression cassette that exceeded
the size actually packaged within the AAV. There are two proposed
mechanisms for this: the fi rst is that recombination occurs between
overlapping homologous genome sequences and the second that
sense and antisense genomes from two AAV vectors anneal with
second strand synthesis occurring from their free 3

′

ends, Fig. 2a .
These potential mechanisms have been used to develop approaches
for large gene therapy using AAV.

′

1. Partially packaged genome approach (Fig. 2a )

Partially packaged genomes containing only one ITR have
been shown by several research groups to transduce host cells
in vitro and in vivo with proof of principle demonstrated in the
muscle [ 59 ] and lung [ 60 ] in addition to the retina [ 55 ]. An over-
lapping dual vector therapy of partially packaged large transgenes
for dysferlinopathies has not only revealed in vivo production of
the full-length 7.7 kb transcript and encoded protein, but this was
also able to promote muscle membrane repair [ 61 ].
2. Overlapping approach (Fig. 2b )

Overlapping approaches contain specifi cally generated frag-
ments of the split coding sequence with a defi ned overlap region in
each vector. The difference to the partially packaged approach is
that two stable AAV genomes are generated with the split trans-
gene contained within two ITRs. This approach has resulted in a
suffi cient cotransduction with subsequent homologous recombi-
nation of the overlapping transgenes to elicit a functional improve-
ment in a mouse model of Duchenne muscular dystrophy [ 62 ].
That this dual vector approach has provided evidence of functional
improvement in other tissue types and models of disease suggests
the potential for such positive effects in the treatment of inherited
retinal dystrophies are also plausible.