Size does matter: overcoming the adeno-associated virus
Terence R Flotte
University of Florida, Gainesville, Florida, USA
Received: 6 June 2000
Revisions requested: 19 June 2000
Revisions received: 20 June 2000
Accepted: 20 June 2000
Published: 5 July 2000
Respir Res 2000, 1:16-18
C Current Science Ltd (Print ISSN 1465-9921; Online ISSN 1465-993X)
Recombinant adeno-associated virus (rAAV) vectors mediate long-term gene transfer
without any known toxicity. The primary limitation of rAAV has been the small size of the
virion (20 nm), which only permits the packaging of 4.7 kilobases (kb) of exogenous DNA,
including the promoter, the polyadenylation signal and any other enhancer elements that
might be desired. Two recent reports (D Duan et al: Nat Med 2000, 6:595-598; Z Yan et al:
Proc Nati Acad Sci USA 2000, 97:6716-6721) have exploited a unique feature of rAAV
genomes, their ability to link together in doublets or strings, to bypass this size limitation. This
technology could improve the chances for successful gene therapy of diseases like cystic
fibrosis or Duchenne muscular dystrophy that lead to significant pulmonary morbidity.
Keywords: adeno-associated virus, cystic fibrosis, gene therapy
Recombinant adeno-associated virus (rAAV) vectors have
some important advantages for gene therapy because
they mediate stable transgene expression in terminally dif-
ferentiated cells without inducing significant inflammatory
toxicity [1-3]. For many years the use of rAAV was some-
what limited by inefficient production methods, but this
problem has recently been addressed by several groups
[4-7], so that now the primary limitation on this system is
its limited effective packaging capacity of approximately
4.7 kb . This has been an important limitation for gene
therapy of cystic fibrosis (CF) , Duchenne muscular
dystrophy, hemophilia A, and other genetic diseases
where the length of the coding sequence approaches this
limit. CF gene therapy is of particular interest to pulmo-
nologists, and the clinical experience with rAAV trials in
CF patients suggests that this agent could be particularly
promising if packaging constraints could be overcome.
Two recent papers from the laboratory of Dr John Engel-
hardt [10,11] describe the exploitation of an unusual
feature of AAV biology to effectively double the packaging
capacity and thus overcome this size constraint.
The mechanism being exploited is the capacity of two dis-
tinct rAAV genomes that happen to infect the same cell to
undergo intermolecular recombination inside the trans-
duced cell nucleus. The discovery of this phenomenon
CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; ITR = inverted terminal repeat; kb = kilobases; rAAV = recombi-
nant adeno-associated virus.
ROLLING CIRCLE REPLICATION
Possible mechanisms for the generation of rAAV concatemers.
stemmed from earlier work on rAAV-derived episomes,
first described in bronchial cells in culture [12,13] and in
the primate airway . The Engelhardt group studied this
phenomenon by using shuttle vectors and found that at
least some of these episomes were circular head-to-tail
concatemers [15,16], which might have been derived
either from rolling circle replication of a single input
genome or from intermolecular recombination of two dis-
tinct input genomes occurring within the palidromic
inverted terminal repeat (ITR) sequences that are found at
each end of the AAV genome (Fig. 1). Recent evidence
favored the latter possibility.
The next step, described in the two recent papers, was to
exploit this feature to circumvent the small packaging
capacity of rAAV. The AAV capsid is only able to hold 5 kb
of single-stranded DNA in most instances. Because a 145
nucleotide stretch of the AAV ITR sequence is required at
each end for the vector DNA to replicate and be pack-
aged, this leaves only about 4.7 kb of effective payload in
each rAAV particle. For genes such as cystic fibrosis
transmembrane conductance regulator (CFTR) (whose
coding sequence approaches 4.5 kb), this leaves little
space for effective promoter, enhancer and polyadenyla-
tion sequences. Indeed, the rAAV-CFTR vector that has
been used in clinical trials in CF patients uses only the
minimal promoter activity of the AAV ITR itself to drive
CFTR expression .
The approach taken by Duan et al  was to package a
'superenhancer', that is, a combination of the potent
simian virus 40 (SV40) and cytomegalovirus immediate
early enhancer elements, in one rAAV vector and a
luciferase reporter gene driven by a small minimal pro-
moter element in the other. They found that either the
SV40 promoter or the intrinsic cryptic promoter activity of
Four possible orientations of products of intermolecular recombination.
When one vector carries the entire transgene and the other an
enhancer, all four are active. When the two vectors carry the two
halves of a single gene-coding region with an intervening intron, only
the first of these is active.
the AAV ITR itself, which had previously been used in
rAAV-CFTR vectors, was sufficient for this purpose. They
found that intermolecular recombination between the two
vectors occurred inside the transduced cells either in vitro
or in vivo. The intermolecular recombination event was
efficient enough to boost transgene expression 200-600-
fold in vivo in muscle.
In a related study, Yan et al used a similar approach to
express long-term functional levels of erythropoietin by
using a two-vector strategy in mouse muscle in vivo .
Intermolecular recombination has actually been used in
slightly different ways in the second paper and in work
described by Sun et al . The latter approach is to
insert the promoter and the first half of the coding
sequence in one rAAV vector, followed by a splice donor
and the upstream half of an intron. The second rAAV
vector contains the downstream half of the intron, the
splice acceptor, the second half of the gene, and the
polyadenylation signal. Once again, this strategy is effi-
cient enough to mediate high-level expression and the
intermolecular junctions are apparently stable enough to
mediate expression for several months in vivo.
Each of these strategies has its advantages and disadvan-
tages. The superenhancer strategy takes maximal advan-
tage of the intermolecular recombination mechanism
because of the position-independent and orientation-
LJ- L -
Respiratory Research Vol 1 No 1 Flotte
independent nature of enhancers. There are four possible
products of an intermolecular recombination event,
because either of the two vectors could be on the 5' end
of the heterodimeric molecule and either segment could
be in either orientation (Fig. 2). With the superenhancer
strategy, all four of these products should be functional for
transgene expression, whereas with the split intron strat-
egy only one of the four would work. The only disadvan-
tage of the enhancer strategy is that the coding sequence
of the gene in question must still fit within a single vector,
whereas the split intron vector expands the packaging
capacity to a greater degree.
In either case, the net effect is that the primary remaining
limitation of rAAV as a gene vector has effectively been
eliminated. As mentioned above, recent preclinical data
indicate that rAAV is safe, efficient, and stable in lung,
muscle, brain, spinal cord, retina, and liver. There still are
obstacles to overcome with regard to the distribution of
the heparan sulfate proteoglycan attachment receptor for
AAV and its co-receptors, the av-P5 integrin or the fibro-
blast growth factor receptor. There is also still some
potential for immune responses, particularly in hosts who
might be entirely naive to the protein being produced.
However, it seems likely that there will be many more rAAV
trials in the coming years. With the newly expanded effec-
tive packaging capacity, the potential future applications
of rAAV are indeed very broad.
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Author's affiliation: Powell Gene Therapy Center, University of
Florida, Gainesville, Florida, USA
Correspondence: Terence R Flotte, Powell Gene Therapy Center,
University of Florida, Box 100266, Gainesville, Florida 32610-0266,
USA. Tel: +1 352 846 2739; fax: +1 352 846 2738;