Size does matter: overcoming the adeno-associated virus packaging limit
0
University of Florida
,
Gainesville, Florida
,
USA
1
Author's affiliation: Powell Gene Therapy Center, University of Florida
,
Gainesville, Florida
,
USA
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 Natl 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.
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Recombinant adeno-associated virus (rAAV) vectors have
some important advantages for gene therapy because
they mediate stable transgene expression in terminally
differentiated cells without inducing significant inflammatory
toxicity [13]. For many years the use of rAAV was
somewhat limited by inefficient production methods, but this
problem has recently been addressed by several groups
[47], so that now the primary limitation on this system is
its limited effective packaging capacity of approximately
4.7 kb [8]. This has been an important limitation for gene
therapy of cystic fibrosis (CF) [9], 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
pulmonologists, 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
Engelhardt [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
distinct rAAV genomes that happen to infect the same cell to
undergo intermolecular recombination inside the
transduced cell nucleus. The discovery of this phenomenon
CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; ITR = inverted terminal repeat; kb = kilobases; rAAV =
recombinant adeno-associated virus.
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 [14]. 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
distinct 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
packaged, 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
polyadenylation sequences. Indeed, the rAAVCFTR 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 [9].
The approach taken by Duan et al [10] 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
promoter 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
rAAVCFTR 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
efficien (...truncated)