Evaluation of Risks Related to the Use of Adeno-Associated Virus-Based Vectors

Transcription

Current Gene Therapy, 2003, 3, 545-565
545
Evaluation of Risks Related to the Use of Adeno-Associated Virus-Based
Vectors
L. Tenenbaum1,2,*, E. Lehtonen1,2 and P.E. Monahan3
1
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire. 2Laboratory of Experimental
Neurosurgery, ULB, Belgium. 3 Gene Therapy Center and Department of Pediatrics, The University of North Carolina at
Chapel Hill, USA
Abstract: Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies. As this vector
is increasingly applied to human clinical trials, it is a priority to evaluate the risks of its use for workers involved in
research and clinical trials as well as for the patients and their descendants.
At high multiplicity of infection, wild-type AAV integrates into human chromosome 19 in ~60% of latently infected cell
lines. However, it has been recently demonstrated that only approximately 1 out of 1000 infectious units can integrate.
The mechanism of this site-specific integration involves AAV Rep proteins which are absent in vectors. Accordingly,
recombinant AAV (rAAV) do not integrate site-specifically. Random integration of vector sequences has been
demonstrated in established cell lines but only in some cases and at low frequency in primary cultures and in vivo. In
contrast, episomal concatemers predominate.Therefore, the risks of insertional mutagenesis and activation of oncogenes
are considered low.
Biodistribution studies in non-human primates after intramuscular, intrabronchial, hepatic artery and subretinal
administration showed low and transient levels of vector DNA in body fluids and distal organs. Analysis of patients body
fluids revealed rAAV sequences in urine, saliva and serum at short-term. Transient shedding into the semen has been
observed after delivery to the hepatic artery. However, motile germ cells seemed refractory to rAAV infection even when
directly exposed to the viral particles, suggesting that the risk of insertion of new genetic material into the germ line is
absent or extremely low.
Risks related to viral capsid-induced inflammation also seem to be absent since immune response is restricted to
generation of antibodies. In contrast, transgene products can elicit both cellular and humoral immune responses,
depending on the nature of the expressed protein and of the route of vector administration.
Finally, a correlation between early abortion as well as male infertility and the presence of wt AAV DNA in the genital
tract has been suggested. Although no causal relationship has been established, this issue stresses the importance of using
rAAV stocks devoid of contaminating replication-competent AAV.
This review comprehensively examines virus integration, biodistribution, immune interactions, and other safety concerns
regarding the wild-type AAV and recombinant AAV vectors.
INTRODUCTION
Recombinant AAV (rAAV)-based vectors efficacy has
been demonstrated in various tissues including muscle [Xiao,
1996], brain [McCown et al., 1996], lung [Conrad et
al.,1996], liver [Snyder et al., 1997] and retina [Ali R, et al.,
1996). Based on successful preclinical studies in animal
models, clinical trials have then been launched in particular
for cystic fibrosis [Wagner et al., 2002], hemophilia [Kay et
al., 2000] and limb girdle muscular dystrophy [Stedman et
al., 2000]. In this context, it is primordial to evaluate the
risks related to the use of rAAV for the environment,
workers involved in research and clinical trials as well as
patients and their offspring. Those include vector shedding
into non-targeted organs and body fluids, vector DNA
integration, immune reactions against viral capsid proteins
and transgene products, inadvertent insertion of genetic
material into the germ line. A clinically oriented review on
safety of AAV vectors has recently appeared [Monahan et
al., 2002].
*Address correspondence to this author at the Institut de Recherche
Interdisciplinaire en Biologie Humaine et Moléculaire, Laboratory of
Experimental Neurosurgery, ULB, Belgium; Fax: 32-2-5554655; E-mail:
litenenb@ulb.ac.be
1566-5232/03 $41.00+.00
In light of the epidemiology of the wild-type (wt) virus,
which, despite ubiquitous exposure in humans, nevertheless
results in no pathology and low immunogenicity, rAAV
vectors are perceived as the most promising system in terms
of safety. This review will first describe the epidemiology
and biology of wt AAV as a foundation for understanding
safety issues related to the recombinant vectors derived from
this virus. However, specific properties of rAAV vectors
cannot be extrapolated from the fundamental knowledge
accumulated on wt AAV since for example, the integration
specificity is different. Thus, safety aspects need to be
addressed by designing specific experiments using
recombinant vectors.
In this review, only aspects that are relevant for the
evaluation of the risks related to the use of AAV vectors will
be presented and discussed. Readers interested in the
technologies developed to construct and produce
recombinant AAV (rAAV) as well as in their potential
applications will find this information in various excellent
reviews [Muzyczka, 1992,1994; Flotte & Carter, 1995;
Carter & Samulski, 2000; Rabinowitz & Samulski, 2000;
Peel & Klein, 2000; Monahan & Samulski, 2000].
© 2003 Bentham Science Publishers Ltd.
546
Current Gene Therapy, 2003, Vol. 3, No. 6
In view of further improvements to the vectors likely to
be required before demonstrating clinical efficacy, additional
biosafety evaluations will be needed. A number of recent
advances in rAAV purification will be discussed, which
support the expectation that biosafety and GMP
requirements will be relatively easily met when producing
clinical lots. Nevertheless, large-scale commercial rAAV
production may still require the use of new cell lines and
helper viruses which will need to be extensively
characterized as to the contamination of the produced stocks
with wt AAV and adenovirus.
WILD-TYPE ADENO-ASSOCIATED VIRUS
Epidemiology
Adeno-associated virus is a member of the Parvoviridae
family, which are among the smallest and structurally
simplest of animal DNA viruses. The family Parvoviridae
has been divided into three genera [Siegl et al., 1985]:
parvoviruses, which are able to replicate in dividing cells of
vertebrates, densoviruses, which multiply in insects and
dependoviruses, which infect a broad range of vertebrates
but usually require a co-infection with an adenovirus for a
productive infection in cell culture.
Because of the frequent association with adenovirus
stocks, the dependoviruses have been called adenoassociated viruses (AAV) [Melnick & McCombs, 1966;
Hoggan et al., 1968]. Different AAV serotypes have been
isolated from avian and mammalian hosts. Human AAVs
were first isolated from the throat or feces of patients
concurrently infected with adenovirus. Initially, four
serotypes were described [Hoggan et al., 1966], types 2 and
3 being the most prevalent types infecting humans, whereas
type 1 and 4 appear to be simian viruses. A fifth serotype has
been isolated from a genital site in 1984 [Bantel-Schaal &
Zur, 1984; Georg-Fries et al., 1984]. AAV5 seems to be rare
and less closely related to the other serotypes than they are
among each other. A sixth serotype exhibiting high
similarities to AAV1, has been isolated as a contaminant
from an adenovirus stock [Rutledge et al., 1998]. Recently,
two novel AAVs designated AAV7 and –8 have been
isolated from nonhuman primates [Gao et al. 2002].
Antibodies directed against capsids from different serotypes,
notably serotypes 2 and 3, usually cross-react as defined by
ELISA and neutralization tests [Erles et al., 1999]. However,
data on crossreactivity between AAV serotypes revisited in
the context of recombinant vectors vary between reports,
probably reflecting differential sensitivity of the assays. For
example, vector readministration studies suggested that
cross-neutralisation between AAV2 and AAV5 is low
[Hildinger et al., 2001] whereas in vitro transduction
inhibition experiments suggested that AAV5 is neutralized
by all other serotypes except 4 [Grimm et al., 2003a]. In
contrast, AAV4 [Grimm et al., 2003] as well as AAV7 and 8
[Gao et al., 2002] are not neutralized by vectors of others
serotypes.
Most adults (85-90% in Belgium and in the USA) are
seropositive for antibodies against AAV [Blacklow et al.,
1967a, 1968b; Luchsinger et al., 1970; Blacklow, 1975].
These data have been recently updated for other countries
[Erles et al., 1999]. Fifty percent of the 20-30 year-olds were
Tenenbaum et al.
found seropositive in Germany, 63% in France, 55% in
Brazil, and 46% in Japan. Seroconversion occurs during
childhood and is usually concomitant with an adenovirus
infection [Blacklow et al., 1967a, 1967b, 1968a, 1968b;
Hoggan et al., 1966; Blacklow et al., 1971]. Among these,
about 30% have neutralizing antibodies [Chirmule et al.,
1999].
Although human infections are common, AAV has never
been implicated as an etiological agent for any disease
[Blacklow et al., 1968, 1971]. The infection appears to be
benign or even beneficial. Indeed, several studies have found
that women with cervical carcinomas are seronegative for
antibodies to AAV [Luchsinger et al., 1970; Georg-Fries et
al., 1984]. These data raise the possibility that AAV might
actually protect against the effects of certain oncogenic
processes [Walz et al., 1997; Raj et al. 2001, Walz et a;.
2002], a hypothesis that is supported by in vitro experiments
in transformed cell lines [Yalkinoglu et al., 1990].
Cryptic infections are also common. AAV can be latent
in human and monkey cells. Up to 20% of the lots of
primary African green monkey kidney cells and 1-2% of
primary lots of human embryonic kidney cells were found to
be naturally latently infected with AAV, which can be
rescued by adenoviral infection [reviewed in Berns &
Bohenzky, 1987].
Despite the lack of evidence for pathogenicity,
correlations have recently been made between:
i). the occurrence of male infertility and the presence of
AAV viral DNA sequences in human semen [Rohde et
al., 1999]
ii). the occurrence of miscarriage and the presence of
infectious AAV in embryonic material as well as in the
cervical epithelium [Tobiasch et al., 1998; Walz et al.,
1997]. A clear association is hard to establish from these
studies, given that co-incident evidence of human
papillomavirus infection is present in most subjects
[Malhomme et al., 1997], and that AAV DNA can be
detected in cervical samples in the majority of women
[Burguete et al., 1999] but is very dependent on
differences in sample collection between studies [Erles et
al., 2001]. In favor of a possible causal role of AAV in
the occurrence of miscarriage, AAV 2 has been shown to
interfere with mouse embryonic development [Botquin et
al., 1994]. Furthermore, a significant correlation has been
established between the presence of AAV DNA in
amnion fluids and premature amniorrhexis and premature
labor [Burguete et al., 1999].
It should be noted that when documented, the
pathogenicity of parvoviruses is related to the Rep proteins,
which are absent in rAAV vectors [Ozawa et al., 1988;
Muzyczka, 1992; Brownstein et al. 1992]. Although vectors
are devoid of a rep-coding sequence, the presence of
biologically active Rep proteins encapsidated in mature
progeny of extensively purified AAV and rAAV particles
has been demonstrated [Kube et al., 1997]. At particle: cell
ratio of 104, growth inhibition of primary human cells has
been described [Kube et al., 1997]. It was shown later that
during productive infection, complexes of Rep proteins and
capsids are formed prior to DNA packaging [King et al.,
Evaluation of Risks Related to the Use of Adeno-Associated
2001]. However, since capsids are rapidly eliminated in
infected cells [Bartlett et al., 2000], Rep-mediated effects, if
any, are probably transient.
Transmission and Reservoir
Grossman and colleagues used PCR to show that AAV
DNA can be detected in blood samples from healthy donors
[Grossman et al., 1992]. However, a higher prevalence of
AAV is found in the genital tract, in the cervical epithelium
[Walz et al., 1997] and in semen [Rohde et al., 1999]. The
concomitant presence in cervical biopsies of human
papilloma virus, which can serve as a helper for AAV
[Hermonat, 1994] furthermore suggests the possibility of
sexual transmission of AAV [Walz et al., 1997].
The concern that AAV might be vertically transmitted
was also raised. In favor of this hypothesis, 27% of amnion
fluid samples scored positive for AAV DNA and 30% of
these contained infectious AAV virions [Burguete et al.,
1999].
Further
supporting
vertical
transmission,
transplacental transmission of avian AAV has been
experimentally demonstrated in mice [Lipps & Mayor, 1980,
1982].
Current Gene Therapy, 2003, Vol. 3, No. 6 547
virus [Walz et al., 1997] and vaccinia virus [Schlehofer et
al., 1986], can also serve as helpers to support AAV
replication. Exposure of several cell lines to agents which
interfere with cellular DNA synthesis, such as chemical
carcinogens, UV irradiation or cycloheximide [Schlehofer et
al., 1983, 1986; Yalkinoglu et al., 1990, 1991; Yakobson et
al., 1989] as well as cell synchronizing treatments
[Yakobson et al., 1987], render the cells permissive for viral
production or at least for AAV macromolecular synthesis.
Therefore, it has been suggested that cellular functions
induced by the helper rather than viral functions are
responsible for the helper effect. The interaction between
AAV and its helpers results in enhancement of AAV
functions whereas helper virus replication is usually
inhibited to some degree [Bantel-Schaal and Zur Hausen,
1988; Heilbronn et al., 1990; Hermonat, 1994].
Recombinant AAV replicates in the presence of wt AAV
and of a helper virus. Therefore, when present in the genital
tract, rAAV is likely to be co-transmitted with AAV. In this
regard, it is very important to carefully evaluate the
biodistribution of vector sequences in relation to the method
of administration (see further).
PRODUCTIVE PATHWAY
The requirement for a co-infection with a helper virus to
establish a productive infection renders AAV naturally
defective. However, human AAV serotypes are able to
replicate in cell cultures derived from different species
provided that the cells are co-infected with a helper virus for
which the cells are permissive (reviewed in [Berns &
Bohenzky, 1987]).
Fig. (2). Adeno-associated virus DNA replication.
Upon entering the cell, the AAV virion particles uncoat in the
nucleus and release their single-stranded DNA genome which is
converted into a T-shape hairpin intermediate. As a result the entire
coding sequence plus the 5’-end inverted terminal repeat (ITR) is
completely replicated. To replicate the 3’-end ITR, a nick is made
by the Rep proteins on the parental strand at the terminal resolution
site (TRS). This nick is then used as a primer to complete
replication of the 3’ ITR. The resulting duplex replicative form
serves as a template for strand displacement synthesis generating a
new double-standed replicative form and a single-stranded AAV
genome which is packaged to produce mature virions.
Solid lines represent parental DNA molecules ; dotted lines
represent newly synthesized DNA molecules. RF represents the
double-stranded replicative form.
LATENCY
Fig. (1). Adeno-associated virus life cycle.
Wild-type AAV can be propagated either in a latent state as an
integrated provirus that can be rescued by superinfection with
adenovirus or by lytic infection in the presence of a co-infecting
adenovirus.
Viruses of different families unrelated to the adenovirus
family, such as herpesvirus [Georg-Fries et al., 1984; Handa
& Carter, 1979; Schlehofer et al., 1986], human papilloma
In the absence of a helper virus, the dependovirion
traffics into the cell nucleus where the DNA is uncoated and
is then integrated into the cell genome [Handa & Shimojo,
1977; Berns et al., 1975]. The genome can be rescued from
the integrated state after superinfection of the cell with a
helper virus [Cheung et al., 1980]. Berns and collaborators
demonstrated that a latent infection could be achieved in cell
culture by simply infecting human cell lines with AAV in the
absence of adenovirus [Berns et al., 1975; Cheung et al.,
1980]. Such culture can remain latently infected for more
548
than 100 passages. At high multiplicities of infection (250
infectious units per cell), AAV has been observed to cause
latent infection in 10-30% of the infected cells. The viral
DNA was found to be integrated into cell DNA as a tandem
repeat of several copies, with the termini of the viral genome
close to the junction with cellular sequences [Kotin & Berns,
1989]. However, it has been recently demonstrated using
real-time PCR that only approximately 1% of the infectious
units are able to integrate site-specifically in HeLa cells
[Hüser et al., 2002].
The cellular sequences flanking AAV provirus were used
as probes to characterize the AAV integration site in latently
infected cell lines. It was shown that in 68% of the cell lines
examined, the provirus was linked to the same cellular
flanking sequences. This sequence, called AAVS1 is
localized on human chromosome 19 [Kotin et al., 1990,
1991; Samulski et al., 1991; reviewed in Samulski, 1993].
The AAVS1 site contains a motif corresponding to a repbinding site (RBS) present in the AAV ITRs [Weitzman et
al., 1994]. It furthermore contains a terminal resolution site
(trs) similar to the site of rep-dependent nicking, which
resolves the AAV replication intermediates during
productive replication (see figure 2). A 33-nucleotide
sequence containing RBS and trs has shown to be sufficient
to mediate site-specific integration in a plasmidic shuttle
vector [Giraud et al., 1994, 1995]. It has been furthermore
shown in a cell-free assay that Rep can form a triple complex
with AAV ITR and the AAVS1 RBS site [Weitzman et al.,
1994] strongly pointing out that Rep proteins function as the
central actors in site-specific integration.
Clones of transformed cells harbouring integrated AAV
sequences show a reduced growth rate and a reduced ability
to form clones in agar [Walz & Schlehofer, 1992] as well as
an increased sensitivity to UV irradiation [Winocour et al.,
1992]. The comparison of cell clones containing integrated
rep+ and rep- vectors suggests that the expression of Rep
proteins is required to observe these inhibitory effects
[Winocour et al., 1992]. Normal cell growth and progression
Tenenbaum et al.
through the cell cycle was also shown to be affected by AAV
infection [Winocour et al., 1988]. However, very high
multiplicities were necessary and no AAV gene expression
was required, since heavily irradiated virus had the same
effect as non-irradiated virus. It should be noted however
that until now, no conclusive evidence for the integration of
wild-type AAV in man has been obtained.
Physical Properties of the Viral Particles
The non-enveloped wt AAV virion has a diameter of 2022 nm. It contains a linear single-stranded DNA genome of
4.7 kb encapsidated within a simple icosahedral capsid
composed solely of three proteins called VP1-3. The 3dimensional structure of the AAV2 capsid has been recently
determined by x-ray crystallography [Xie et al., 2002]. AAV
DNA strands of both polarities are packaged into separate
virions and with equal frequency. The AAV particles are
stable in a wide pH range between 3 and 9 and are resistant
to heating at 56°C for at least 1 h. The particles can be
dissociated by exposure to either a combination of papain
and ionic detergents or an alkaline pH (reviewed in [Berns &
Bohenzky, 1987]).
Since the production of wt AAV depends on the presence
of a helper virus, purification requires the separation of AAV
and helper virus particles. This is usually achieved using
cesium chloride gradients. The buoyant density of infectious
AAV particles is 1.39-1.42 g/cm3. The particle: infectivity
ratio of these particles is usually equal to or lower than
1:100. Since the density of adenovirus is 1.37 g/cm3, the
separation between the two viruses is incomplete and
therefore AAV preparations have to be heated at 56°C for 30
min to 1 hr in order to inactivate the residual adenoviruses.
In addition, infectious virus particles of lower (from 1.32
g/cm3) and higher (up to 1.45 g/cm3) densities can be
harvested from the gradients [de la Maza &Carter, 1980].
The lighter particles have been shown to be empty particles.
It has been proposed that the intermediate densities (1.321.39 g/cm3) correspond to partial genomes and that these
Fig. (3). Adeno-associated virus genome and open reading frames.
The AAV genome has been arbitrarily divided into 100 map units. The position of the inverted terminal repeats is indicated by bold lines
flanking the genome. The positions of the three AAV promoters (p5, p19 and p40) are shown. All mRNA terminate at a common
polyadenylation signal located at map unit 96. The p5 and p19 promoters direct the synthesis of the non-structural Rep proteins whereas p40
directs the synthesis the Cap proteins which constitute the viral capsid. The position of the introns is shown.
function as defective interfering particles. The high-density
particles (1.45 g/cm3) have a DNA and protein composition
similar to the infectious particles. However their particle:
infectivity ratio is drastically (10-20-fold) higher [de la Maza
&Carter, 1980].
AAV-BASED VECTORS
Genetic structure (see Fig. 3)
The isolation of two different molecular clones of AAV2
by ligation of the double-stranded form of the virus in
pBR322 plasmids [Laughlin et al., 1983; Samulski et al.,
1982, 1983] has opened doors for the genetic analysis of the
virus [Hermonat et al., 1984]. The cloned genome is
infectious when transfected into a permissive cell, i.e. the
viral genes are expressed and the viral genome is rescued
from the plasmid, replicated and then encapsidated to form
infectious virus [Samulski et al., 1983]. There are two large
open reading frames in the wt AAV genome; the left one
codes for the « Rep » regulatory proteins and the right one
for the « Cap » structural proteins. The coding region is
flanked on both sides by non-coding inverted terminal
repeats (ITRs) of 145 bp. Genetic analysis of AAV mutants
indicated that the only cis-acting sequences required for
AAV rescue from bacterial plasmids, DNA replication and
encapsidation are the AAV ITRs. Consequently, 95% of the
wt AAV genome can be deleted and substituted by foreign
DNA [review: Muzyczka, 1992] to create recombinant
vectors.
Risks Related to Production Strategies
Traditional protocols for producing rAAV vectors consist
of a cotransfection into adenovirus-infected 293 cells of a
rAAV vector plasmid and a packaging plasmid providing in
trans the AAV rep and cap genes [Samulski et al., 1989,
Snyder et al., 1996]. Consequently, rAAV preparations were
contaminated with adenoviral particles [Snyder et al., 1997].
Furthermore, homologous and nonhomologous recombination events between the vector and the complementing
plasmid generate wild-type or wild-type-like replicationcompetent AAV (rcAAV) particles [Allen et al., 1997].
CREATION
OF
REPLICATION-COMPETENT
PARTICLES BY RECOMBINATION
Although wt AAV is incapable of replicating without a
helper virus, its presence is highly undesirable for clinical
applications (see above: epidemiology). The presence of
residual adenovirus (or other helper viruses) or even
marginal amounts of helper virus proteins is similarly
undesirable in view of their potential toxicity and
immunogenicity. The vector plasmids in current use retain
only the 145bp ITRs from the AAV genome.
The vector plasmids in current used helper plasmids
encoding rep and cap genes have no homology with the
vector plasmids, in order to minimize recombination events
between the helper and vector plasmids giving rise to
replication-competent plasmids. Note that, in this context,
« replication-competent » refers to DNA molecules that,
upon adenovirus infection, can give rise to replicative centers
in permissive cell lines (see further: titration assays) without
addition of wt AAV. However, even using pairs of
vector/helper plasmids harboring no homology (see Figure
4) the resulting viral stocks have been shown to be
contaminated up to 10% with rcAAV [Wang et al., 1998].
Analysis of the virus’ replicative DNA revealed that the
contaminating genomes were not authentic wild-type but
AAV-like sequences resulting from non-homologous
recombination between AAV ITRs in the vector plasmid and
AAV sequences in the helper plasmid. The identification of a
10 bp sequence in the ITRs present at the recombination
junctions led to the development of strategies for the
elimination of wt-like genomes. Recombination events
producing rcAAV-like viral particles even occur when a
heterologous promoter replacing the endogenous p5
promoter [Allen et al., 1997] is used.
CONTAMINATION WITH ADENOVIRUS
Adenoviral genes required for rAAV production have
been identified as: E1a, E1b, E2a, E4orf6 and VA RNA. In
all production strategies described above, 293 cells (which
express the adenovirus E1a) are used as a producer cell line
and adenovirus as a helper virus. Adenovirus is then heatinactivated at 56°C during the purification process.
However, residual adenoviral proteins are usually still
present in the final preparation.
Helper adenovirus can be totally eliminated thanks to
novel plasmids, which contain only those adenovirus genes
required for the AAV helper effects, except those already
present in 293 cells [Grimm et al., 1998; Xiao et al., 1998].
In the system developed by Xiao et al., a triple transfection
is performed with the AAV vector plasmid, a plasmid
containing rep and cap genes, and a « mini-adeno » plasmid
containing the E2a, E4 and Va RNA adenoviral genes.
In the system designed by Grimm et al., the AAV rep
and cap genes have been included in a single « mini-adeno »
plasmid under the control of the dexamethasone-inducible
promoter of MMTV (mouse mammary tumor virus) thus
creating pDG (see figure 5). Titration by in situ focus
hybridization assay [Yakobson et al., 1989] shows that
laboratory-scale rAAV prepared by means of this plasmid
are characterized by a complete absence of replicationcompetent virions from either AAV or adenovirus (our data).
Recently, similar helper virus-free production systems for
AAV vectors of serotypes 1 to 6 have been developed
[Grimm et al., 2003b].
However, transient transfection is difficult to adapt for
the purpose of large-scale commercial preparation of clinical
lots. Therefore, producer cell lines are being developed,
which contain both the vector and the rep and cap genes and
therefore do not require any transfection step but rather
infection with a helper virus. These cell lines have been
difficult to isolate, due to the toxicity of Rep proteins.
Therefore, they usually are rep-inducible [Clark et al., 1995;
Inoue & Russell, 1998]. However, these systems rely on
overexpression of viral proteins and the titers obtained are
usually not higher than those of the transfection methods.
Furthermore, production still depends on infection with wt
adenovirus and wild-type-like AAV contaminants do appear.
550
Tenenbaum et al.
Fig. (4). Production of recombinant AAV virus by the co-transfection/adenovirus infection method.
Recombinant AAV virus is produced by co-transfection of HEK-293 cells with i) the vector plasmid harboring the transgene and regulatory
sequences bracketed by AAV non-coding inverted terminal repeats (ITRs) and ii) a helper plasmid containing the AAV coding region (for
example : pIM45 ; Mc Carty et al.,1994). One day posttransfection, the cells are infected with adenovirus at a multiplicity of 3 particleforming units per cell. Forty-eight hours after infection, the cells are lysed by cycles of freeze-thawing and the lysate is purified by 2 rounds
of CsCl gradients (Snyder et al., 1996 ; Tenenbaum et al., 1999). This procedure gives rise to recombinant viral preparations which are
contaminated with replication-competent AAV particles, originating from recombination events between the vector plasmid and the helper
plasmid, and residual adenovirus uncompletely separated by the CsCl gradients. The latter can be heat-inactivated at 56°C resulting in rAAV
preparations contaminated by adenoviral proteins and nucleic acids.
An alternative strategy uses replication-defective herpes
simplex virus (HSV) to deliver the rep and cap genes and
thus generate a single infectious helper, the HSV-rep-cap
[Conway et al., 1999]. This recombinant HSV virus is used
to infect cell lines harboring the rAAV construct. Cell lines,
which do not complement the HSV vector replication defect,
produce rAAV without generating additional HSV-rep-cap.
The same recombinant HSV can be used for serial passages
of rAAV virus in order to increase the titer of the viral
preparation. Residual HSV-rep-cap helper is heat-inactivated
at 55°C.
Attempts to isolate adenoviral vectors expressing rep and
cap genes have failed until recently, probably due to the
inhibitory effect of Rep proteins on adenovirus replication. A
complete adenovirus-based method in which all components
necessary for rAAV production were delivered by
adenovirus has been proposed [Zhang & Li, 2001]. This new
method relies on the inducible expression of rep and cap,
which are under the control of a tetracycline-responsive
promoter.
ii) a second adenoviral vector containing the AAV coding
sequence with the tetracyline-responsive promoter
instead of the endogenous p5 and
iii) a third adenoviral vector containing the tetracycline
transactivator under the control of the constitutive CMV
promoter.
Also this method allows a serial passaging of the rAAV
virus resulting in higher titers. The rAAV stocks obtained by
means of this method were devoid of wild-type virions as
evaluated by Southern blot analysis and replication-center
assay (less than one active wild-type particle in 108 active
rAAV
particles).
However,
potential
adenovirus
contamination, inherent to the adenoviral vectors technology
has not been ruled out. Heparin-sulfate-based columns (see
below), which have been shown to potentially completely
eliminate adenovirus contamination [Zolotuhkin et al.,
1999], could be used to purify rAAV obtained by this
method. The complete absence of contaminants when
producing rAAV with either inducible adenovirus-rep-cap or
HSV-rep-cap remains yet to be demonstrated.
The production scheme consists in a triple infection with:
i) an adenoviral vector containing the transgene and
regulatory sequences bracketed by AAV ITRs ,
PURITY
The discovery of the heparan sulfate proteoglycan
(HSPG) as a receptor for AAV 2 [Summersford & Samulski,
1998] led to the use of heparin columns [Zolotuhkin et al.,
Fig. (5). Production of recombinant AAV virus by the co-transfection method.
Recombinant AAV virus is produced by co-transfection of HEK-293 or HEK-293T cells with i) the vector plasmid harboring the transgene
and regulatory sequences bracketed by AAV non-coding inverted terminal repeats (ITRs) and ii) a helper adenoviral vector plasmid
containing the AAV coding region and the adenoviral genes required for the adenovirus helper effect (Grimm et al. , 1998). Alternatively,
helper adenoviral genes and AAV coding region can be provided using 2 separate plasmids in triple co-transfections (Xiao et al. , 1998).
Forty-eight to 50 hours after transfection, the cells are lysed by cycles of freeze-thawing and the lysate is further purified by iodixanol
gradient combined with heparin agarose chromatography (Zolotukhin et al., 1999). Using this procedure at the laboratory scale, rAAV stocks
devoid of detectable replication-competent viruses are obtained (Lehtonen et al., unpublished).
1999] instead of CsCl gradients for virus purification,
allowing a higher recovery as well as a more scalable
process usable in GMP production. Since at least some
alternative serotypes do not use HSPG as a receptor
[Rabinowitz et al., 2002], other purification techniques will
need to be developed. Recently, AAV4 and AAV5 have
been shown to bind sialic acid and consequently mucin (a
sialic acid polymer) agarose columns were proposed to
purify rAAV5 [Auricchio et al., 2001]. The use of
immunoaffinity columns using an antibody specifically
recognizing viral capsids is also very promising [Grimm et
al., 1998]. Purity is an important issue for the evaluation of
safety. Using the new affinity columns combined to HPLC,
the resultant drug is considerably cleaner than that obtained
using previous purification methods that were largely based
on less specific virus properties such as density and size
[Zolotukhin et al., 1999]. Use of improved ion exchange
HPLC without affinity columns however allows purification
of vectors based on different serotypes with the uniform
technique [Kaludov et al., 2002; Drittanti et al., 2001].
When purifying rAAV5, a similar or slightly higher
(depending on the particular vector construct) virus yield in
terms of transducing units was obtained using the ion
exchange HPLC [Kaludov et al., 2002 ] versus mucin
column [Auricchio et al., 2001] method. Furthermore, the
use of HPLC in combination with a high molecular weight
retention filter step [Kaludov et al., 2002] resulted in rAAV5
with a higher ratio of full versus empty particles than
previously described purification methods.
Risks Related to the Titration Method
Classically, wt AAV and adenovirus are used to titer
infectious rAAV particles in replicative center assays
[Yakobson et al., 1989]. The issue of high risks of laboratory
contamination with wild-type viruses is raised by this
method. In particular, contaminant wt AAV cannot be
eliminated from contaminated cell lines or viral stocks.
Therefore, it is advisable to use alternative replicative assays
such as co-infection with rAAV and adenovirus of cell lines
carrying integrated rep-cap sequences [Liu et al., 1999]. This
constitutes an improvement in the safety of AAV
technology, since it does not require the presence of wildtype AAV in the laboratory, thus reducing the risk of
contamination of cell lines, rAAV and adenovirus stocks.
Other titration methods include quantification of the
number of rAAV genomes by dot-blot [Samulski et al.,
1989; {Potter et al., 2002], and quantification of the number
of viral capsids by ELISA [Grimm & Kleinschmidt, 1999].
[Grimm & Kleinschmidt, 1999]. However, the latter methods
do not address the biological functionality of recombinant
viral particles. Of particular interest, are anti-capsid
antibodies detecting only DNA-containing viral particles.
Risks Related to Integration
The fate of recombinant AAV DNA sequences after in
vivo delivery is a crucial issue for the risk:benefit evaluation
of gene therapy protocols.
552
Tenenbaum et al.
Fig. (6). Titration of rAAV stocks by in situ focus hybridization assay.
The in situ focus hybridization assay (Yakobson et al., 1989) is a biological test revealing genomes with replication-competent ITRs.
Subconfluent HeLa cells were infected with 3 µl (A), 10 µl (B) or 30 µl (B) of a 10,000-fold diluted rAAV stock, in the presence of excesses
of wild-type AAV and adenovirus. Twenty-eight hours after infection, the cell monolayer was transferred to a nitrocellulose filter, alkali
denatured and hybridized with a 32P-labeled DNA probe directed to the transgene. Foci of cells synthesizing rAAV DNA were vizualized
after autoradiography. Titers were evaluated by counting the number of autoradiographic spots (A,27 ; B, 118; C,280) and expressed as
infectious units per ml (1 x 108 I.U./ml).
The property of site-specific integration is not retained by
rep- vectors [Yang et al., 1997; Recchia et al., 1999].
Instead, persistent expression of vector sequences may occur
from randomly integrated sequences as well as from
extrachromosomal (episomal) sequences [Flotte et al., 1994;
Nakai et al., 2001]. In vitro, in non-dividing cells, some
vector sequences integrate [Wu et al., 1998]. This is also the
case -although in a limited fashion- in transformed cell lines
[Cheung et al., 1980; Yang et al., 1997; Duan et al., 1997;
Miller et al., 2002]. In vivo, several lines of evidence suggest
that most early transgene expression derives from episomal
vectors [Flotte et al., 1994; Goodman et al., 1994] which
consist of circular concatemers [Yang et al., 1999; VincentLacaze et al. 1999; Schnepp, et al., 2003]. This evidence
shows that, at least in non-dividing cells, integration is
neither required for transgene expression, nor for stability of
the vector. At long-term, however, vector sequences were
often found associated with high molecular weight DNA
[Xiao et al., 1996; Clark et al., 1997; Wu et al., 1998; Malik,
et al., 2000]. Whether these represent genomic DNA
contaning integrated vector sequences or large episomes has
been a matter of debate. In one case, PCR products obtained
using one primer in repetitive genomic sequences and one
primer in the vector provided convincing evidence for
integration in the brain [Wu et al., 1998]. However, the
frequency of occurrence of such integrants has not been
evaluated in this study. In another study, integrated vector
sequences were detected at low frequency in the liver
[Nakai, et al., 2001]. Recently, the absence of
chromosomally-integrated
vector
sequences
after
intramuscular injection in mice has been unequivocally
demonstrated [Schnepp et al., 2003]. In the latter case,
expression from skeletal muscle has been correlated with the
widespread presence of episomal concatemers. Previous
studies in primary cultures of muscle cells have already
suggested that high molecular weight vector-containing
DNA could mainly represent large viral concatemers rather
that integrated forms [Malik et al. 2000 ]. It should be noted
that, when demonstrated, in vivo rAAV DNA integration
seems to be a low frequency event. The limits of sensitivity
of experimental systems used to examine in vivo integration
in skeletal muscle and liver allowed the conclusion that at
least 99.5% (skeletal muscle) and at least 90% (liver) of
persisting vector DNA was not integrated [Schnepp et al.,
2003; Nakai et al. ,1999; Nakai et al., 2001]. In conclusion,
the majority of the persistent rAAV sequences observed in
animal studies are likely to represent large episomal
concatemers. However, whether the concommitant
occurrence of integrated vector sequences represents a safety
concern is still a matter of debate.
The ITR sequences have been shown to contain a weak
transcriptional promoter [Flotte et al., 1993; Haberman et al.,
2000]. When vectors are integrating, the risk of activation or
interruption of cellular genes is raised. In fact, viruses that do
not require integration as a step in the wild-type life cycle
may still carry a risk of DNA recombination with the host
chromosomal DNA. For example, adenovirus as well as
early-generation adenovirus vector DNA sequences have
been shown to integrate into primary and transformed cell
lines [Schagen et al., 2000; Harui et al., 1999]. Such
illegitimate recombination might be reduced using “gutless”
adenovirus vectors, but argues for long-term follow-up of all
patients involved in gene therapy trials with any vector.
A number of investigations of rAAV vector integration in
transformed cell lines [Yang et al., 1997; Rutledge and
Russel 1997; Miller et al., 2002; Huser et al.2002] or in mice
(which do not carry sequences homologous to the human
AAVS1 integration site) [Nakai et al., 1999; Nakai et al.
2003] suggest that rAAV’s “random” integration favors
transcriptionally active regions of the host genome
[Rivadeneira et al. 1998; Nakai et al. 2003], that is to say,
within the introns and exons of genes . Rearrangements of
vector sequences in the process of integration occur mainly
in the ITR and flanking regions, while changes in transgene
sequences are rare. Great variability in the extent of
chromosomal sequence rearrangements, duplications or short
deletions is seen with vector integration in these
experimental systems. In two different model systems, the
observed deletions of cellular gene sequences were usually
2bp to <300 bp and occured in active genes [Russell, et al.
2002; Nakai et al. 2003]. Most systems designed to examine
chromosomal effects have employed immortalized cells or
tissues induced to actively proliferate; the possibility remains
that AAV integrates preferentially into chromosome regions
with existing DNA strand breaks, rather than creating breaks
[Miller et al., 2002].The importance of such variable nonhomologous recombination events following in vivo vector
integration in normal human cells or tissues is an unresolved
safety concern.
Although activation or interruption of cellular genes
represent real possibilities associated with randomly
integrating viral gene transfer vectors, in practice neither the
inactivation of a tumor suppressor gene nor activation of an
oncogene leading to tumor formation has ever been
documented using helper-free AAV stocks [Schagen et al.,
2000]. The chance of an individual integration event leading
to detrimental mutagenesis is likely to be very small
(especially when considered in comparison to the
spontaneous mutation rate of a single base in the mammalian
genome [Cole and Skopek, 1994; Kazazian, 1999; Kazazian
and Goodier, 2002 ; Schnepp, et al., 2003]. It appears that
the integration of non-Rep-containing vectors occurs in a
small minority of rAAV infections, if at all, in tissues that
are not actively dividing. Nevertheless, concern regarding
the safety of rAAV vectors was raised by a disturbing
finding in a recent AAV-mediated gene therapy experiment
utilizing a mouse model of the lysosomal storage disease
mucopolysaccharidosis Type VII (MPS VII) [Donsante et
al., 2001]. This lysosomal storage disease in humans,
resulting from deficient activity of β-glucuronidase, leads to
multi-organ disease and premature death, with the clinical
picture closely modeled in the MPS VII mouse. Following
treatment as newborns with an AAV2 vector encoding the βglucuronidase gene, MPS VII mice were observed over 18
months with striking clinical improvement, prolonged
survival, and no apparent toxicity. However, over an 18month period at least 6 out of 59 treated MPS VII mice
developed metastatic liver tumors (hepatocellular carcinoma
and/or angiosarcoma). The development of liver cancers was
particularly unsettling in light of the fact that previous longterm studies in this extensively characterized strain of mice
using other therapeutic approaches (e.g. bone marrow
transplantation and enzyme replacement therapy) failed to
uncover any predisposition of MPS VII mice towards tumor
formation. To investigate rAAV-mediated insertional
mutagenesis as the causative event in hepatic tumor
formation, the number of AAV genomes per cell in the
tumor samples was examined. The simplest model of
mutagenesis-mediated tumor formation from a clonal
malignant proliferation predicts that one copy of the vector
genome per host genome should be detected per cell in the
tumor. Instead, it was discovered that the number of AAV
genomes per cell in the tumor samples ranged from
undetectable (less than one copy per 1000 diploid genomes)
to 0.113. Other hypotheses might explain the tumor
formation in this model: dramatic overexpression of the
therapeutic protein in a small percentage of cells, chronic
occupation of the mannose-6-phosphate/IGII receptor by the
therapeutic protein, delivery of the viral vector during an
early developmental period (day 1-2 of life). These
hypotheses are being investigated [Vogler et al., 2003].
It should be noted that no other group has observed
tumor formation following rAAV administration. However,
most of the systematic studies designed to determine the
toxicity of rAAV have been performed for less than one year
and the rate of tumor formation in the MPS VII study was
relatively slow (5 of the 6 tumors appeared in animals one
year old or greater). Positive outcomes for the field from this
unexpected treatment complication should include a greater
emphasis on appropriate design of long-term toxicity studies,
where possible in the relevant animal models, and an
openness to sharing pre-clinical adverse event reporting
among investigators [Flotte et al., 2001;Verma 2001].
It is possible that mutations in the ITRs could be
designed to minimize undesirable properties of the ITR, such
as promotion of transcriptional activity and rescue from
latency [Young and Samulski, 2001].
It should be noted that the trs and RBS sites are retained
in the vectors and that Rep proteins have been shown to
promote site-specific integration of recombinant AAV when
provided in trans [Lamartina et al., 1998; Palombo et al.,
1998; Recchia et al., 1999]. In these systems, integration is
both more efficient and predictable.
PHYSICAL PROPERTIES
VIRAL PARTICLES
OF
RECOMBINANT
Risks Related to the AAV Capsid
Recombinant AAV particles are similar to wt AAV
particles. However, rAAV preparations contain different
types of particles having different stabilities [Kube et al.,
1997]. AAV capsids elicit a humoral immune response, and
the percentage of empty capsids in a given rAAV preparation
is important to evaluate. Titration of viral particles by capsid
ELISA provides this information [Grimm & Kleinschmidt,
1999]. Different production methods yield different amounts
of empty particles. The total particle titer of rAAV is equal
to or slightly lower than that of wt AAV stocks. In contrast,
the number of DNA-containing particles is usually markedly
lower for rAAV (ratio of physical to DNA-containing
particles of 60:1). The number of infectious or transducing
particles is even lower (less than 0.1% of the assembled viral
particles). These findings are consistent with electron
microscopy analysis of rAAV2 stocks in which the majority
of observed rAAV particles were found to be empty capsids
[Grimm et al., 1998]. A recently developed purification
method based on HPLC and high molecular weight retention
filter centrifugation (Centriplus) allows generation of
rAAV2, rAAV4 and rAAV5 viral preparations containing
90% of full particles as estimated by the comparison of the
quantity of DNA and proteins and confirmed by electron
microscopy [Kaludov et al., 2002 ].
VIRUS-CELL INTERACTIONS
DNA microarrays have recently been used to screen
changes in cellular mRNA levels resulting from infection
554
with wt AAV or rAAV [Stillwell and Samulski, 2001,
Stillwell and Samulski, 2003]. A relatively limited number
of cellular genes demonstrated changes in their expression
pattern (38 of 5600 mRNA showed a >4-fold increase or
decrease) in the primary diploid cells tested (IM4-90 primary
lung fibroblasts). The majority of changes were decreases of
expression in genes related to cell cycle regulation, DNA
replication and kinesin-like motor proteins. Interestingly,
similar changes were seen after infection with empty AAV
capsids. This suggests that the AAV particles themselves are
able to set in motion molecular events leading to decreased
cell proliferation, which are potentially conducive to the
establishment of latency. These trends offer some molecular
insight into the antiproliferative effect of wt AAV infection
that has been described phenotypically and correlated with
apparent antitumor effect in some [Raj et al., 2001; Smith et
al., 2001; Walz and Schlehöfer, 1992] but not all [Strickler
et al., 1999; Odunsi et al., 2000] studies.
IMMUNE RESPONSES TO THE CAPSID
Recombinant AAV vectors do not contain any viral
genes, leaving the transgene product and the virus capsid as
the only source of foreign antigen. No cellular immune
response to the VP proteins has been described following
rAAV-mediated gene transfer to muscle, lung, brain, or
retina [Joos et al., 1998; Fisher et al., 1997; Xiao et al.,
1996; Hernandez et al., 1999; Herzog et al., 1997; Song et
al., 1998; Conrad et al., 1996; Lo et al., 1999; Mastakov et
al., 2002; Anandet al., 2002]. A single report showing an
innate cellular immune response following high doses of
rAAV2 administered to the mouse liver has recently been
published [Zaiss et al., 2002] The response was markedly
truncated, however, with chemokine (e.g. TNF-α)
upregulation demonstrable at 1 h postinfection but returned
to baseline at 6 h postinfection (next time point examined).
This study is complicated by the use of AAV vector
produced with live Ad, with potential contamination by Ad
capsid proteins, which are immunostimulatory independent
of infectious capacity [Molinier-Frenkel et al., 2002; Stilwell
JL, McCarty DM, et al. 2003]. In the DNA microarray
analysis (see Virus-Cell Interactions, above), cells exposed
in vitro to wt AAV, rAAV, or empty AAV capsid particles
demonstrated no changes or decreases in mRNA for cellular
inflammatory mediators or stress response genes [Stilwell et
al. 2001; Stillwell et al. 2003].
Nevertheless antibodies directed against the AAV capsid
proteins are frequently generated, and a subset of these can
block subsequent vector infectivity. These neutralizing
antibodies can significantly reduce the efficacy of vector readministration in some but not all settings [Moskalenko et
al., 2000; Beck et al., 1999; Halbert et al., 1998]. In animal
studies of rAAV administration to the muscle or liver, vector
serotype-specific
anti-AAV
neutralizing
antibodies
consistently have developed. These neutralizing antibodies
have eliminated the efficacy of rAAV readministration
following liver-directed [Moskalenko et al., 2000; Xiao W,
et al. 2000] and some intramuscular [Xiao X, et al. 2000;
Manning, et al., 1998] approaches, but only blunted
transduction in others [Chirmule et al., 2000; Fisher et al.,
1997]. However, anti-AAV neutralizing antibody formation
is reportedly transient and low titer or absent following
Tenenbaum et al.
delivery to the brain [Lo et al., 1999; Mastakov et al., 2002]
and retina [Anand et al., 2000; Anand et al., 2002] and
repeat administration will likely be efficacious. The effect of
pre-existing neutralizing antibodies on transduction of
animal lung has been inconsistent [Halbert, et al., 1998;
Beck et al. 1999], and emphasizes the recognition that
animal models will be limited in predicting the response by
the human immune system to human and primate serotype
capsids, as well as to transgene products (see below).
Eight subjects in a human clinical trial of intramuscularly
administered rAAV2 carrying the gene for coagulation factor
IX demonstrated increases in anti-AAV2 neutralizing
antibody titers of one to three logs at 1 and at 6 months after
treatment [Manno et al., 2003]. Gene delivery in this phase I
trial is difficult to quantify, but did not appear to correlate
with pre-existing or treatment-related neutralizing antibody
titers. In a clinical trial for cystic fibrosis, no changes in
serum neutralizing antibody titers were seen after delivery of
low doses (108 AAV2 particles) to the maxillary sinus
[Wagner et al., 2002; Wagner et al., 1998]. Aerosolized or
bronchoscopic delivery of the same vector to the lung at 1011
to 1013 particles led to serum neutralizing antibodies in some
patients in two trials. No correlation with degree of gene
transfer could be determined [Aitken et al. 2001].
It is not clear whether naturally-acquired immunity to wt
AAV will impact rAAV gene delivery. While pre-existing
antibodies to AAV2 are found in up to 90% of humans,
neutralizing antibodies occur in a much smaller subset
(~30%) and only 5% of one series of patients tested had
peripheral blood lymphocytes that proliferated in response to
AAV antigens [Chirmule et al., 1999]. The use of alternative
serotypes, more especially those isolated from non-human
primates such as AAV4, AAV7 and AAV8 [Gao et al.,
2002], may overcome the obstacle of seroprevalent antiAAV2 antibodies. One cited “risk” of participation in Phase
I/II clinical trials using viral gene vectors is that participants,
after developing antibody responses to the AAV vector, will
lose the chance for later treatment with improved AAV
vectors. It appears likely that treatment strategies in some
sites (e.g. subretinal) and using some vector modifications
(e.g. serotype capsid switch) [Beck et al., 1999; Halbert et
al. 2000] will permit successful re-administration of AAV
vectors.
HUMORAL AND CELLULAR IMMUNE RESPONSES
TO THE TRANSGENE PRODUCT
Although long-term expression of transgenes after AAV
delivery has been achieved with a variety of transgenes,
cellular and humoral immune responses against the encoded
gene product have arisen in some applications [Manning et
al., 1997; Monahan et al., 1998; Herzog et al. 1999; Zhang
et al., 2000; Brockstedt et al., 1999; Sarukhan et al. 2001a;
Yuasa et al., 2002 ]. Therefore, while rAAV vectors carry an
advantage of relatively low immunogenicity, they do not
appear to bear a universal mechanism of immune evasion.
Likewise, when immune responses to the transgene product
have been characterized, a variety of factors contribute.
These include the route of vector administration, the cellular
localization, tissue-specific expression and intrinsic
immunogenicity of the transgene product (including whether
derived from cDNA of homologous or heterologous species),
and host factors such as the major histocompatibility
complex haplotype as well as underlying gene defects of the
host. Two reports in 1996 demonstrated that after
intramuscular rAAV injection in mice, persistent expression
of intracellular E. coli β-galactosidase [Xiao et al., 1996] or
secreted human erythropoietin [Kessler et al., 1996] was
achieved without cellular immune response. The rAAV β-gal
transduced cells persisted even as a cytotoxic T lymphocyte
response selectively eliminated cells in the same muscle that
were concurrently transduced with rAd-β-gal. A partial
explanation came from investigations showing that rAAV2
only directly transduce professional antigen presenting cells
(mature dendritic cells) at a very low rate [Jooss et al.,
1998]. Subsequent investigations have shown that immature
dendritic cells (DC) [Zhang et al., 2000] and DC precursors,
on the other hand, can be transduced by rAAV and
effectively participate in activation of T cells. This
differential transduction has been exploited for a rAAVbased vaccine strategy [Ponnazhagan et al., 2001].
Recognition that DC transduction may contribute to
elimination of transgene expression supports strategies using
tissue-specific promoters to avoid transgene expression
outside the specific target cells [Cordier et al., 2001 ].
In the absence of direct transduction of antigenpresenting cells (APCs), the likely mechanism for immune
responses to rAAV-delivered transgenic proteins is crosspresentation of antigen by APCs [Kurts, 2000; Sarukhan et
al., 2001a; Sarukhan et al. 2001b]. A number of factors that
have been implicated in anti-transgene immune responses
may contribute to this mechanism, including site of
localization of the transgenic protein, the route and
mechanics of the vector administration, and the level of
transgene produced. For instance, in contrast to the persistent
expression of β-galactosidase protein (LacZ) in muscle
described above, a modified rAAV intramuscular vector
expressing LacZ retargeted from the cytoplasm to the cell
surface resulted in the induction of LacZ -specific cellular
immune responses and target cell destruction [Sarukhan et
al., 2001b]. Accordingly, rAAV-lacZ vectors that elicit no
immune responses when expressing intracellularly in normal
mice, have nevertheless generated immune responses when
administered to dystrophin-deficient mice with poor cell
membrane integrity [Yuasa, et al. 2002]. Regarding
expression of the factor IX transgene (F.IX), humoral
immune responses against F.IX have been stimulated more
consistently following muscle transduction than liver
transduction [Ge et al., 2001]. The fact that transgenic factor
IX is trapped locally in muscle by collagen IV binding, in
contrast to efficient intravascular secretion from the liver,
may facilitate F.IX antigen cross-presentation via cellular
localization, similar to the LacZ example.
Levels of antigens in peripheral tissues must be high in
order to facilitate priming of naïve CD8+ T cells by crosspresentation [Kurts et al. 1998]. In the example of I.M.
rAAV for hemophilia gene therapy, in addition to cellsurface localization of the transgenic protein, the absolute
level of transgene expression achieved has been implicated
in antibody formation in some investigations [Herzog et al.
2002], although not in others [Chao and Walsh, 2001]. In
addition, more weakly-expressed protein may activate naïve
T cells if the target tissue is destroyed or inflammation
elicited, effectively presenting a danger signal in conjunction
with the novel transgene [Kurts et al., 1998; Gallucci et al.,
1999; Matzinger et al., 2002]. The underlying myocyte
degeneration of muscular dystrophy may provide danger
signals that promote cross-presentation and the loss of
expression from AAV-γ-sarcoglycan or AAV-lacZ vectors
reported in mouse models of muscular dystrophy [Cordier et
al., 2001 ; Yuasa, et al., 2002 ]. As another example, the
intramuscular route of administration leads to greater
bleeding and inflammation than the intravenous route.
Intramuscular factor IX gene therapy may produce a unique
combination of factors all favoring immune priming by
cross-presentation particular to this high-profile rAAV
application but not others [Song et al., 2002].
Unexpectedly, the recent human clinical trial of
intrahepatic delivery of rAAV/human factor IX has provided
evidence of both apparent efficacy and immune responses,
not observed in the human intramuscular trial for hemophilia
B. [Kay et al., 2002] A subject in the highest dose cohort (2
x 1012 vector genomes/kg; revised dose estimate) achieved
factor IX levels of >10% for three weeks, followed at week 4
postinfection by an elevation of liver transaminases,
indicating hepatocytes injury. While the liver studies
returned to normal over six weeks, circulating factor IX also
fell to <1%, in a pattern suggesting that a significant portion
of transduced cells were eliminated. The role and
mechanisms of immune response to vector and transgene in
this subject are under investigation. An additional subject,
treated at the same dose and with a more modest and
transient factor IX expression, has not shown evidence of
toxicity or immune response. These examples underscore the
limitations of predicting human immune responses from
available models, and support efforts to coordinate
development of uniform models and assays to mimic human
responses [The Second Cabo Gene Therapy Working Group,
2002].
Biodistribution
The biodistribution of a recombinant vector should be
evaluated independently for each different route of
administration. In addition, differential transduction by
alternative AAV serotypes of individual cell types within
organs have begun to be described. Differences in the global
patterns of biodistribution with the alternative serotypes, e.g.
after systemic administration, are also being mapped [Grimm
et al., 2003a; Monahan et al., 2003]. Recombinant AAV2
vector DNA scatter beyond the site of administration has
been examined in several animal and 2 clinical studies (see
Table). After intramuscular delivery of rAAV2 in a dog
model of hemophilia, PCR tissue analysis revealed vector
sequences in injected muscle tissue and adjacent lymph
nodes, without dissemination to other organs (including
gonads) [Monahan et al., 1998; Chao et al., 1999].
Nevertheless, dogs are not a natural host forAAVs, and
non-human primate pharmacokinetic and safety studies will
likely be more predictive of results in humans. Studies in
macaques demonstrated that rAAV2 therapy to the muscle
results in detectable vector sequences in serum for up to 6
days [Favre et al., 2001]. Tissue examined at 18 months after
556
Tenenbaum et al.
intramuscular injection showed PCR-detected rAAV in liver,
peripheral blood mononuclear cells and scattered lymph
node sites. No macaques had detectable vector DNA in the
gonads, a finding consistent with another recent trial of I.M.
rAAV in baboons [Song et al., 2002]. Following
intrabronchial administration of AAV2 particles to the lungs
of rhesus macaques, vector sequences were detected in
peripheral blood up to 14 days after treatment, with half of
the animals testing positive for vector DNA in distal organs,
albeit at quite low levels (<0.01% of cells). Again, no vector
DNA was detected in the gonads [Conrad et al., 1996]. A
cohort of macaques treated to the liver via the hepatic artery
also shed rAAV sequences in urine and plasma for up to six
days; tissue analysis of a single animal revealed
hepatosplenic vector distribution without gonadal spread
[Nathwani et al., 2002]
A recent study attempts to model in several animal
species the risk of rAAV2 gene transfer to male germ cells
[Arruda et al., 2001]. rAAV vector spread to gonadal tissue,
semen and spermatogonia in mice and rabbits following
Table.
intramuscular administration, as well as in rats and dogs after
hepatic artery administration were examined. At doses used
in human clinical trials, vector sequences were not detected
in cells from semen of rabbits or dogs by PCR or Southern
blot following intramuscular injections but could be detected
in testicular tissue. Fluorescence in situ hybridization
analysis of the testicular tissue from day 7 postinfection
demonstrated vector sequences localized to the testicular
basement membrane and interstitial space but not within
germ cells. In addition, in vitro attempts to directly infect
isolated murine spermatogonia with a rAAV vector failed.
The investigators concluded that the risk of inadvertent
transduction of male germ cells is very low[Arruda et al.,
2001]. The same authors have recently reported additional
analyses in rabbits after intravenous rAAV [Arruda et al.,
2003] and rats after hepatic artery rAAV [Arruda et al. 2001]
doses ranging from 1011 to 1013 vector genomes/kg (doses
similar to those used in a human clinical trial)[Kay et al.,
2003]. A dose-dependent increase in PCR positive vector
sequences was seen in semen samples; at no timepoint could
infectious AAV particles be recovered from the semen
PCR Analysis of Body Fluids for rAAV Vector Sequences
1 day
Route of delivery
Fluid
3-6 days
> Week 1
Species
(subjects testing positive/subjects analyzed)
Intramuscular
Subretinal
Serum
Rabbit1,
Macaque2
Human3,¶
0/3
0/8
1/8 (Remains + week12)
7/8
1/6
2/8
0/8
0/8
0/8
Macaque2
Human3,¶
Feces
Macaque2
4/8
2/8
0/8
2
Saliva
Macaque
Human3,¶
3/8
6/8
0/8
N.A.
0/8
1/8 (Latest +: week 2)
Lacrymal
Macaque2
2/8
2/8
0/8
Nasal
Macaque2
3/8
4/8
0/8
Dog1,¶
Rabbit1, ¶
Human3, ¶
[First sample at 1.5 months]
Semen
Serum
Dog4
Macaque
5
0/4
0/3
0/7
0/3
0/7
N.A.
0/7
6/6
6/6
6/6 (Latest +: 25 days)
1/2
1/2
1/2 (Latest +: 16 days)
Plasma/Serum
Rabbit
Macaque6
Human7, 8
27/27
4/4
N.A.
N.A.
0/4
4/5 (Latest +: 10 weeks)
Urine
Macaque6
2/4
0/4
Salary
6
1/4
0/4
N.A.
N.A.
0/3
6/6 (Latest+: 14 weeks)
Macaque
1
Semen
1
N.A.
5/8
N.A.
Urine
4
Hepatic delivery
3/3
8/8
6/6
Dog
Human7, 8
Arruda, et al., 2001; 2Favre, et al., 2001; 3Manno, et al., 2003; 4Weber, et al., 2003; 5Arruda, et al., 2003; 6Nathwani, et al., 2002; 7Kay, et al., 2002
In these studies, intramuscular injections were performed with ultrasound guidance to avoid inadvertent intravascular delivery. No signal detectable in motile sperm.
samples [Arruda et al., 2003]. No formal study of rAAV
tranduction of female germ cells has been performed.
The most relevant studies of viral shedding are those
from human clinical trials. Cystic fibrosis transmembrane
conductance regulator (CFTR) vectors have been detected
after airway delivery of rAAV2 in peripheral blood and
sputum for 1 day and 1 week, respectively [Aitken et al.,
2001]. Screening by PCR after rAAV2 intramuscular gene
therapy for coagulation factor F.IX deficiency revealed
vector sequences in the serum during the first 7 days after
treatment, in the urine, saliva during the first 48 h [Manno et
al., 2003], but no positive semen samples at any timepoint.
Nevertheless, the first six subjects of a trial delivering a
similar rAAV2/F.IX vector via the hepatic artery produced
positive semen samples. Upon separate analysis of sperm
and semen, however, fractions containing motile germ cells
tested negative for transferred vector sequences. The low
level PCR signal cleared within weeks in all the subjects but
remained positive for greater than 3 months in one subject
[Kay 2003].
An additional concern regarding “distribution” of rAAV
vectors has to do with the mode of persistence of AAV
sequences through the mechanism of latency. In tissue
culture systems, AAV vector genomes can be rescued from
latency and replicate upon infection with wt AAV and helper
adenovirus. The risk of rescue of recombinant vectors after
in vivo gene transfer is not established, but could
theoretically result in undesirable promiscuous spread of
vector beyond the targeted organ (e.g. to antigen-presenting
cells) or even to the treated subject’s environment. Afione, et
al. attempted to rescue vector from AAV/CFTR-treated
macaques in three separate rescue models. rAAV was
delivered to the lower lobe of the lung or to the nose and a
high dose of wt AAV and adenovirus was administered
through the nose of the animal. Vector shedding was not
detected; only by administering large doses of wt AAV to
the lower lung lobe followed by subsequent rAAV to the
same area and nasal Ad could rAAV sequences be made to
rescue [Afione et al., 1996]. Similarly, Favre, et al., after
establishing that vector sequences persist in peripheral blood
mononuclear cells of I.M.-treated macaques, were unable to
produce rescue of vector sequences from the blood cells after
coincident wt AAV and either Ad or herpesvirus helper
infection [Favre, et al., 2001]. Therefore, the risk of
horizontal dissemination of rAAV appears to be quite low
but worth close monitoring in human trials. Recombinant
AAV theoretically could be transmitted to sexual partners or
to embryos together with wt AAV and a helper (e.g. HPV)
(see above: Epidemiology). As experience in human trials
accumulates, transgenes that are likely to be dangerous for
the development of embryos should be used with caution and
the use of barrier contraceptives by trial participants is
advisable.
Risks of Dissemination
Several members of the Parvoviridae family were shown
to be efficiently transmitted either directly through contacts
between infected and healthy subjects or indirectly between
a healthy subject and the contaminated environment. This is
due to the high survival rate of these viruses outside the host,
probably because of the high stability of the capsid [Berthier
et al., 2000; Myiamoto et al., 2000; Shamir et al., 2001;
Truyen et al., 1998]. For example, contaminating parvovirus
B19 can survive heat treatment and other specific virusreduction techniques in the processing of plasma-derived
coagulation factors, and lead to seroconversion or
symptomatic infection in factor recipients. It is therefore
likely that rAAVs with similarly stable capsids [Xie et al.
2002] can remain biologically active in the environment for
long periods of time. Recombinant AAV vectors have
retained infectivity and transduction capacity at room
temperature for at least a month after simple dessication
[Monahan et al., 2000] or lyophilization [Croyle, et al.
2001].
CONTROL OF TRANSGENE EXPRESSION
For clinical applications it is often desirable to limit
transgene expression to a defined time frame and/or to
precisely control its level. In addition, ideally, in order to
avoid undesirable effects, such as long-term toxicity or
immune response, transgene expression should be restricted
to the particular cell type targeted.
The main limitation of rAAV vector technology resides
in their low cloning capacity (4.5 kb). The impact of the size
limitation is likely to be drastic for efficient regulation of
gene expression, e.g. with inducible promoter systems or the
use of large cellular tissue-specific promoters (for example,
see above: immunological aspects), and therefore affects the
emergence of vectors that meet safety requirements.
Tight regulation of transgene expression has been usually
achieved using 2 separate vectors [Rivera et al. 1999;
Rendahl et al., 1998; McGee Sanftner et al., 2001]. This
strategy works only if the multiplicity of infection is high
enough to ensure a sufficient level of co-infection with both
vectors after in vivo delivery and therefore the production of
very high-titer rAAV is required. Initial attempts to design
single vectors achieving tight regulation resulted in a high
level of residual gene expression in the non-induced state,
due to transcriptional interferences [Haberman et al., 1998;
Rivera et al., 1999]. After “leakiness” of a tetracyclineregulated AAV system was noted, a specific 37 nt region of
the AAV ITR was identified that weakly promotes gene
expression. [Haberman et al. 2000; Flotte, et al. 1993]
Whether this weak promoter activity will complicate AAV
approaches that require tight regulation is an unresolved
safety consideration. Additionally, humoral and cellular
immune responses directed against the tetracyclinedependent transactivator have complicated one intramuscular
rAAV approach.[Favre et al., 2002] More recently,
regulatable single-cassette rAAV vectors with relatively low
background have been obtained using a bi-directional
tetracycline-responsible promoter and either the tetracycline
transactivator resulting in a tetracycline-repressible system
[Fitzimmons et al., 2001], or the reverse transactivator
resulting in a tetracycline-inducible [Chtarto et al., 2003]
system.
Tissue-specific gene expression is also difficult to
achieve given the large size of cellular promoters. Vectors
using truncated promoters which nevertheless retain tissue-
558
specificity are currently under investigaion (Yuasa et al.,
2002).
New AAV-based Vectors
With current developments in capsid modification
methods such as mutagenesis [Rabinowitz & Samulski,
2000; Wu et al., 2000], peptide insertion [Girod et al., 1999],
immunological retargeting [Bartlett et al., 1999], and vectors
based on serotypes 1, 3-8 [Rutledge et al., 1998; Chao et al.,
2000; Xiao et al., 1999; Rabinowitz et al., 2002; Zabner et
al., 2000; Gao et al. 2002] several of the issues discussed
above - notably biodistribution - will have to be re-evaluated.
The conversion of the single-stranded viral genome to
double-stranded form is a limiting step in rAAV-mediated
transduction causing a delay in transduction in some cell
types and a drastically reduced transduction efficiency in
others [Ferrari et al., 1997; Fisher et al., 1997]. An
interesting approach to overcome this problem is the use of
self-complementary or “double-stranded” (ds) AAV vectors
[McCarty et al., 2001; McCarty et al., 2003 (in press
citation); Xiao X et al., 2003 (in press citation)]. The
occurrence of dimeric intermediate replicative forms during
the productive cycle is exploited by designing vectors having
half the wild-type AAV size. Indeed, during the production
of recombinant virus, the dimeric forms whose size is within
the limits of packaging capacity can be encapsidated. These
dimeric forms consist of two inverted complementary copies
of single-stranded DNA which can fold to form a
functionally double-stranded vector DNA. Upon infection of
a target cell with self-complementary vectors, onset of
transgene expression is immediate since these vectors do not
require cellular factors to undergo second-strand synthesis,
thus generating a double-stranded template for transcription.
Self-complementary vectors may allow efficient delivery of
genes smaller than 2.2 kb using a reduced viral number as
compared to full-length vectors. This strategy is likely to
improve the safety of rAAV vectors by reducing the risk of
vector scatter and immune response to the capsid proteins.
CONCLUSIONS AND RECOMMENDATIONS
Risk Assessment
Recombinant AAV biology is distinct from that of wt
AAV. The vectors contain no viral coding sequence. In
particular, they do not express Rep proteins which play a key
role not only for DNA replication but also for site-specific
integration and cellular growth inhibitory effects. Therefore,
the biosafety assessment of rAAV must rely on studies
performed directly with the vectors in relevant animal and
cellular models. Recently accumulated data enlighten the
specific biological properties of rAAV such as integration
specificity and efficiency [Flotte et al., 1994; Goodman et
al., 1994; Duan et al., 1997; Yang et al., 1997; Rutledge and
Russell, 1997; Wu et al., 1998; Yang et al., 1999; Nakai et
al., 1999; Young and Samulski, 2001; Nakai et al., 2001;
Miller et al., 2002; Wu et al. 1998 ; {Schnepp et al. , 2003;
Nakai et al., 2003], immunogenicity [Xiao et al., 1996;
Fisher et al., 1997; Herzog et al., 1997; Manning et al.,
1997; Halbert et al., 1998; Jooss et al., 1998; Song et al.,
1998; Beck et al., 1999; Hernandez et al., 1999; Brockstedt
et al., 1999; Moskalenko et al., 2000; Sarukhan et al., 2001a;
Tenenbaum et al.
Sarukhan et al., 2001b; Ge et al., 2001; Zaiss et al., 2002]
and biodistribution [Manno et al., 2003] [Conrad et al.,
1996; Afione et al., 1996; Monahan et al., 1998; Kay et al.,
2000; Favre et al., 2001; Arruda et al., 2001; Aitken et al.,
2001; Marshall, 2001].
In cell culture, rAAV genome can be rescued and
replicated by superinfection with wtAAV and a helper virus.
However, in vivo rescue experiments failed [Favre et al.,
2001] except in one case in which very large doses of
wtAAV and adenovirus were administered in a particular
setting [Afione et al., 1996]. Thus, adventitious
contamination of workers or material in the laboratory is
likely to be restricted to the amount of rAAV initially
present. Nevertheless, given the high stability of AAV
particles, the presence of even very low amounts of rAAV
should be identified and adequate decontamination should be
undertaken (see below). More especially, rAAV harboring
potentially harmful inserts should be manipulated with
caution (at least as class 2 viruses), since they could have
undesirable effects even at low doses when infecting human
through uncontrolled routes.
Pre-existing immunity to AAV in a large proportion of
the human population might complicate the use of rAAV
vectors derived from serotypes isolated from human
samples. However, only 30% of the population have
neutralizing antibodies and in only 5% of patients,
proliferation of peripheral blood lymphocytes was observed
in response to stimulation by AAV antigens [Chirmule et al.,
1999]. Evidence of gene transfer has now been demonstrated
in a human clinical trial despite measurable neutralizing
antibody titers [Manno et al., 2003].
Insertional mutagenesis, more especially when leading to
tumorigenicity, is a theoretical concern for any gene therapy
vector, and in particular for integrative ones. The ITR
sequences seem to have a structure with the potential for
recombination structure even in the absence of Rep proteins
[Yang et al., 1999]. Although integration of vector
sequences into the cellular genome seems to occur
preferentially into transcriptionally active regions
[Rivadeneira et al., 1998], except in one particular case, no
formation of tumor has been observed in rAAV-mediated
therapy in animals, even after long-term follow-up [Flotte et
al., 2002]. Following treatment of newborn β-glucuronidasedeficient mice with a rAAV2 vector encoding βglucuronidase, 6 out of 59 mice developed liver tumors after
18 months [Donsante et al., 2001]. Given the very low
number of rAAV copies per cell in these tumors, insertional
mutagenesis does not seem to be an explanation for their
occurrence. In view of the increasing number of animal
studies performed at long-term and showing no tumor
occurrence, it is more likely that the tumors that arose in
mucopolysaccharosis model are related to a specific aspect
of this model. However, the risk of insertional mutagenesis
should be taken into account and further evaluated.
Preliminary data on patients undergoing gene therapy
trials showed that, when semen scored positive rAAV for
DNA sequences, these were not contained in the motile
sperm cells, suggesting that vertical transmission of
transgene sequences through inadvertent infection of male
germ cells is not likely to occur [Kay et al., 2000; 2002].
Lack of infection of murine spermatogonia in vitro also
supports this conclusion [Arruda et al., 2001]. However, this
issue requires further and more extensive studies in human.
In particular, it will be also important to study the infectivity
of rAAV vectors for female germ cells.
Finally, recently suggested correlations between i) the
occurrence of male infertility and the presence of wt AAV
DNA in human semen [Rohde et al., 1999] and ii) the
occurrence of miscarriage and the presence of infectious wt
AAV virus in embryonic material as well as in the cervical
epithelium [Tobiasch et al., 1994; Walz et al., 1997] prompt
further evaluation of the classification of wt AAV, as a class
1 agent.These data argue against the use of wt AAV in the
laboratory (e.g. when titering rAAV stocks) and for the use
of a production method which does not give rise to
replication-competent particles.
Laboratory Confinement in Research and Development
Facilities
The high stability of rAAV particles demands the
complete inactivation of all contaminated material before its
removal from the P2 laboratory. Alkaline solutions at pH
higher than 9 can be used for that purpose. When the use of
alkali is impossible (for example for centrifugation buckets),
material should be autoclaved.
Concomitant presence of wild-type and recombinant
viruses in the same laminar flow or in the same incubator
should be totally excluded. Whenever possible, plasmids or
cell lines containing necessary genes from wild-type AAV
and/or adenovirus should be used instead of infectious virus
in production [Clark et al., 1995; Xiao et al., 1998; Grimm et
al., 1998] and titration [Liu et al., 1999; Grimm and
Kleinschmidt, 1999] procedures.
Recombinant virus production methods allowing
virtually no contamination with wild-type viruses should be
preferred. At the laboratory scale, the use of the pDG
plasmid expressing all necessary AAV and adenoviral genes
[Grimm et al., 1998] meets these requirements.
Alternatively, the 3 plasmids system using separate
adenoviral and rep/cap plasmids [Xiao et al., 1998] can be
used with similar results and safety profile. New tools
developed for large-scale production are still to be
completely evaluated. Stocks manufactured using the current
producer cell lines or recombinant viral helpers should go
through a thorough quality control in order to screen for the
presence of wild-type viral contaminants.
Purity is an important issue for the evaluation of safety.
The chosen purification methods should yield a final product
with low amounts of contaminant proteins and nucleic acids
[Zolotukhin et al., 1999]. A purification strategy using a
ligand-affinity column, originally developed following the
identification of heparin sulfate proteoglycan (HSPG) as the
primary receptor for AAV2 [Summersford and Samulski,
1998], greatly reduces contamination with cellular proteins
and potentially toxic reagents such as cesium chloride used
in early steps. With the recognition of sialic acid as a
primary receptor for AAV5 [Kaludov et al., 2001], a singlestep affinity column rich in sialic acid has been developed
and greatly increases the purity of this alternative serotype
vector [Auricchio et al., 2001]. Alternatively, ion exchange
high- performance liquid chromatography (HPLC]-based
purification for AAV2, AAV4, and AAV5 have recently
been developed [Kaludov et al. 2002]. Furthermore,
protocols yielding a high ratio of full-to-empty particles
should be preferred in order to reduce the risk of immune
response against the capsid [Kaludov et al., 2002].
Finally, the stability of AAV viral particles (which allow
a final filtration step) [Drittanti et al., 2001] - as compared to
retroviral and lentiviral vectors - reduces the need for a
highly sterile environment, thereby lowering the costs of
GMP production. Thus, rAAV with higher purity can be
obtained at lower costs, making it more likely that biosafety
requirements will be relatively easily met when producing
clinical lots.
Recent evaluations of rAAV vector scatter to body fluids
in animal and human studies can be used to develop safety
precautions minimizing rAAV vector contamination in the
laboratory setting. Ideally, animals used for evaluation of
rAAV vectors should be housed in a BL2 laboratory
separated from the rest of the BL2 facility. Although this
precaution may not always be practical, it minimizes 1) the
risk of experiments being confounded by adventitious
viruses with potential AAV helper functions, and 2) the risk
of facility contamination by the very stable rAAV particles.
While available PCR analyses for persistent AAV genomes
in treated subjects (see Table 1) certainly overestimate the
persistence of infectious particles, a prudent approach is to
autoclave bedding, bottles and cages from the initial 1-2
weeks after vector administration. Workers should wear
resistant gloves and adsorption masks for small particles
when manipulating the animals and animal blood in
particular.
Additional Safety Concerns for Clinical Trials
Contaminating viral or cellular proteins in the stocks
should be avoid in view of immunological reactions (for
example, adenoviral proteins are very immunogenic). If a
residual presence, even at low doses, were to be assessed in
the stocks, extensive dose-response toxicity and
immunological tests on laboratory animals should be
performed before defining a safety-compatible dose
threshold.
The clinical protocol of administration should be
designed in order to use as a delivery site an organ in which
the immune response is reduced or absent (for example,
antibodies directed against the transgene product are present
at lower titer after intra-hepatic than intra-muscular
administration).
The presence of both AAV and HPV in the uterine tissue
raises the question of potential sexual and/or transplacental
co-transmission of wild-type and recombinant AAV. The
presence of rAAV in the genital tract should be carefully
analyzed. Patients involved in gene therapy trials should be
advised to use barrier contraceptives and should be
submitted to regular testing of blood, urine, feces and sperm
samples.
560
Epidemiologic data for the incidence and prevalence of
neutralizing antibodies for different AAV serotypes (nonserotype 2) for different human populations and at different
ages needs to be established. The age or developmental stage
of the patient may be a useful parameter for consideration in
tailoring efficacious gene therapy. Pre-existing neutralizing
antibodies for different AAV serotypes should be screened
for in individual patient populations (or individual patients)
and in the future, when new vectors will be available for the
clinics, the gene therapy protocol (serotype of the vector,
capsid modification, route of administration) could be
adapted according to the immunological status of the
patients.
Transduction efficiency of the current vectors based on
serotype 2 is generally lower than those achieved using
adenoviral or lentiviral vectors. A higher efficiency would
increase safety by reducing the amount of recombinant virus
needed to obtain clinically relevant effects. The use of
stronger or less attenuated promoters such as the
CMV/chicken β-actin hybrid promoter (CBA) [Xu et al.
2001; Klein et al., 2002] and regulatory elements stabilizing
the messenger RNA, such as the woodchuck hepatitis
posttranscriptional element (WPRE) [Loeb et al., 1999]
result in higher efficiencies. For optimal safety in some
applications, however, the use of tissue-specific promoters is
preferable to stronger ubiquitous promoters, e.g. when
ectopic transgene expression in antigen-presenting cells may
evoke immune responses against the gene or genetransduced cells. Furthermore, vectors derived from other
AAV serotypes might provide a more efficient transduction
of cell types that are poorly transduced by rAAV2 vectors
[Chao et al., 2000; Davidson et al., 2000; Zabner et al.,
2000; Gao, et al. 2002.]. However, each new serotype will
require a separate evaluation of its biosafety profile.
ACKNOWLEDGEMENTS
We thank Dr Francis Dupont and Myriam Sneyers for
stimulating discussions.
E.L. is a recipient of a fellowship from the Belgian
National Research Foundation (F.N.R.S./ Télévie). PEM is
supported by NIH K08 HL03960-01 and R01 HL65404-01.
This work was also supported by grants of the Belgian
National Research Foundation (FNRS-FRSM) n° 3.4565.98,
n° 3.4619.00 and n°3.4501.02 and by grants from “Société
Générale de Belgique” and from “Bruxelles-Capitale”.
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