Hum Genet (2006) 119: 571–603
DOI 10.1007/s00439-006-0165-6
R EV IE W
Kenneth H. Warrington Jr Æ Roland W. Herzog
Treatment of human disease by adeno-associated viral gene transfer
Received: 16 January 2006 / Accepted: 28 February 2006 / Published online: 13 April 2006

Springer-Verlag 2006
Abstract During the past decade, in vivo administration
of viral gene transfer vectors for treatment of numerous
human diseases has been brought from bench to bedside
in the form of clinical trials, mostly aimed at establishing
the safety of the protocol. In preclinical studies in animal
models of human disease, adeno-associated viral (AAV)
vectors have emerged as a favored gene transfer system
for this approach. These vectors are derived from a
replication-deficient, non-pathogenic parvovirus with a
single-stranded DNA genome. Efficient gene transfer to
numerous target cells and tissues has been described.
AAV is particularly efficient in transduction of non-
dividing cells, and the vector genome persists predomi-
nantly in episomal forms. Substantial correction, and in
some instances complete cure, of genetic disease has
been obtained in animal models of hemophilia, lyso-
somal storage disorders, retinal diseases, disorders of the
central nervous system, and other diseases. Therapeutic
expression often lasted for months to years. Treatments
of genetic disorders, cancer, and other acquired diseases
are summarized in this review. Vector development,
results in animals, early clinical experience, as well as
potential hurdles and challenges are discussed.
Introduction
Gene therapy is a promising novel approach toward
treatment of human disease. A gene encoding a thera-
peutic peptide, protein, or RNA is transferred into a
K. H. Warrington Jr Æ R. W. Herzog
Cellular and Molecular Therapy, Department of Pediatrics,
University of Florida, Gainesville, FL, USA
R. W. Herzog (&)
University of Florida, 13706 Innovation Drive,
Room 101, Alachua, FL 32615-9586, USA
E-mail: rherzog@ufl.edu
Tel.: +1-386-4626139
Fax: +1-386-4624099
particular target tissue in vivo or a cell ex vivo (followed
by implantation of the genetically engineered cells into
the body). Genetic disease can be corrected through gene
replacement therapy based on introduction of a func-
tional copy of the defective gene. Transfer of ribozymes
or RNAi can reduce levels of an undesired endogenous
gene product, the accumulation of which is caused by a
dominant mutation. Other therapeutic genes may aug-
ment metabolic pathways or immune responses such as
in the case of anticancer therapy. The currently most
effective means of gene transfer are provided by genetic
engineering of recombinant viruses, which have evolved
to efficiently infect mammalian cells.
Adeno-associated viral (AAV) vectors are based on a
non-pathogenic, replication deficient member of the
parvovirus family with a 4.7 kb single-stranded DNA
genome (Flotte 2004). These vectors are capable of
transducing a variety of tissues and cell types in vivo
with the potential of directing long-term expression,
while showing lower immunogenicity than many other
vector systems. Thus, AAV-mediated gene transfer is
under investigation for treatment of a large number of
diseases. The wild-type virus encodes rep and cap genes,
which are required for viral replication and synthesis of
capsid proteins, respectively. Through a combination of
alternative translation start and splicing sites, the small
genome is able to express four rep and three cap gene
products (Fig. 1). Humans are likely infected through
the respiratory tract. Infection of a cell in the absence of
a helper virus results in the onset of the latent cycle, in
which the virus may integrate with preference to a spe-
cific site (AAVS1) on human chromosome 19 based on
in vitro studies (Kotin et al. 1992; Linden et al. 1996a, b;
Philpott et al. 2002). This site-specific integration ap-
pears to be less frequently observed in vivo, where it
persists mostly in episomal form (Chen et al. 2005b;
Schnepp et al. 2005). In the presence of a helper virus
(such as adenovirus, herpes virus, or others), the AAV
genome is excised and replicated through intermediate
double-stranded forms. The rep gene products and se-
quences in the inverted terminal repeats (145 bp ITRs,
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Fig. 1 Genetic map for wild-type and recombinant AAV. The wild-
type genome is comprised of the left REP open reading frame that
expresses four replication proteins (Rep 78, 68, 52, and 40) from
two promoters (p5 and p19) using alternative splicing; and the right
CAP open reading frame that expresses three capsid proteins (VP1,
2, and 3) from one promoter (p40) using alternative splicing and
which flank the genome) are critical in this process. The
vector is devoid of viral coding sequences, i.e., rep and
cap have been replaced with an expression cassette for
the therapeutic gene (Fig. 1) (Samulski et al. 1982,
1987). While the ITR has been shown to possess mild
promoter activity (Flotte et al. 1993), for maximum
levels of transgene expression, the cassette typically in-
cludes a promoter/enhancer combination, a small intron
sequence, the cDNA of the therapeutic gene, and a
polyadenylation signal (Fig. 1). Other post-transcrip-
tional regulatory elements can be used in addition or
instead of an intron. Production of the vector is often
accomplished by triple transfection of a human embry-
onic kidney cell line (HEK-293), with one plasmid con-
taining the AAV vector sequence (the expression cassette
flanked by the ITRs), the second plasmid encoding rep
and cap genes, and the third providing adenoviral helper
functions (E4, VA, and E2A, with E1 being provided by
the HEK-293 genome) (Liu et al. 2003; Xiao et al. 1998).
Alternative production systems are based on rep/cap
expressing producer cells (derived from HELA cells), on
insect cells infected with baculovirus, or on use of Her-
pes helper virus (Clark et al. 1995; Conway et al. 1997,
1999; Urabe et al. 2002). Both + and strand vector
genomes are packaged into viral particles comprised of
capsid proteins VP1, VP2, and VP3, which are present at
a ratio of approximately 1:1:10. The first and most
extensively studied AAV vector system is based on
serotype 2. AAV-2 vectors can infect a broad range of
cell types in vitro and have been shown to efficiently
transduce non-dividing target cells such as muscle fibers,
hepatocytes, and neurons in vivo. This often resulted in
long-term expression without destructive cellular im-
mune responses against virally transduced cells, which
had been seen for other viral vectors. In fact, AAV-2 was
found to be substantially less likely to cause innate and
adaptive immune responses to its viral capsid or to the
transgene product than previously favored adenoviral
vectors, which also efficiently transduce cells in vivo
start codon usage. The entire genome is flanked by the cis-acting
terminal repeats (TR). The REP and CAP open reading frames are
replaced by a transgene expression cassette (promoter, intron,
transgene, and pA) in the recombinant AAV vector genome. This
TR-flanked cassette is packaged into AAV vectors by providing the
Rep and Cap proteins along with helper virus functions in trans
(Fisher et al. 1997; Jooss et al. 1998; Xiao et al. 1996;
Zaiss et al. 2002). This can in part be attributed to AAV-
2’s low efficiency in transducing professional antigen
presenting cells (e.g., macrophages or dendritic cells) in
vivo (Sarukhan et al. 2001). Since these observations had
been made in the mid to late 1990s, AAV-2 has been
extensively evaluated as a vector platform to deliver
genes in vivo, i.e., through injecting the vector directly
into the mammalian body, in order to treat genetic
disease, hoping that a single administration of vector
could result in long-lasting therapeutic expression in a
particular target tissue. However, depending on the
disease and target cell, transgene expression did not al-
ways achieve desired levels. More recently, it was found
that alternate serotypes differ in their tissue tropism and
ability to transduce different cell types. Initially, six
known serotypes (AAV1-6) were investigated for gene
transfer as a function of the route of vector adminis-
tration (intramuscular, systemically, etc.) by pseudo-
typing or ‘‘trans-packaging’’ of the AAV-2 genome into
capsid from a different serotype (Davidson et al. 2000;
Rabinowitz et al. 2002; Rutledge et al. 1998; Xiao et al.
1999). The vector genome is flanked by AAV-2 ITR
sequences, and the packaging plasmid retains AAV-2
rep, but encodes cap sequences from a different serotype.
AAV-2, -3, and -5 are believed to be of human and
AAV-1 and -4 of simian origin. AAV-6 represents a
recombination (possibly in the laboratory) between
AAV-1 and AAV-2. A large proportion of the human
population has been exposed to AAV-2 (50–96% sero-
positivity, depending on the study and assay used), with
at least one-third of patients having neutralizing anti-
body to AAV-2 (Chirmule et al. 1999; Erles et al. 1999).
Seropositivity is most likely the result of respiratory
infection and increases with age during childhood.
Since 2002, a large number of novel serotypes have
been isolated from non-human primates, human tissue,
and other sources, mostly using PCR-based techniques
(Gao et al. 2002, 2003). Some of these have shown

remarkable levels of gene transfer to various tissues,
albeit that the safety of some of these approaches re-
quires further study. Our knowledge about the cellular
receptors of AAV vectors is limited. Heparan sulfate
proteoglycan (HSPG) is the primary receptor for AAV-
2, which can utilize aVb5 integrin, fibroblast growth
factor receptor-1, or hepatocyte growth factor receptor
as co-receptors for internalization after binding to the
cell surface (Kashiwakura et al. 2005; Qing et al. 1999;
Summerford and Samulski 1998). AAV-5 requires a-2,3-
N-linked sialic acid for efficient binding on the cell sur-
face and utilizes platelet-derived growth factor receptors
a and b as co-receptors (Di Pasquale et al. 2003; Walters
et al. 2001). While AAV-4 has been shown to depend on
alpha-2,3-O-linked sialic acid for cell binding, the co-
receptor has yet to be identified (Kaludov et al. 2001).
Use of receptors by other serotypes is not known yet.
When considering AAV as a gene transfer vector, one
has to be aware of potential limitations for this vector
system. The vector has a packaging limit of 5 kb,
which makes it unsuitable for larger cDNAs. There are
several strategies being developed to overcome this limit,
some based on dual vector systems, which will be dis-
cussed in the following text. Gene transfer to a particular
target cell may be limited at one of the several steps in
vector trafficking (Fig. 2). First, the vector particle needs
to bind to a cellular receptor and then be internalized
through interactions with a co-receptor. After viral
entry, the vector particle has to escape the endosome in
order to avoid degradation, traffic through the cyto-
plasm to the nucleus, and translocate to the nucleus.
After or during entry into the nucleus, the particle has to
uncoat to release the vector genome. The single-stranded
DNA has to be converted into a double-stranded form
for transgene expression and stability. Depending on the
serotype and target cell, all of these steps during trans-
duction can be a serious limitation for gene transfer.
Second-strand synthesis and annealing between com-
plementary strands from two different vector genomes
have been proposed as mechanisms for formation of a
double-stranded genome (Ferrari et al. 1996; Nakai et al.
2000a). Intra- and intermolecular recombination leads
to the formation of circular forms and concatemers, with
mostly episomal persistence and low frequency of inte-
gration (only 5% of stably transduced hepatocytes
have integrated vector genomes, perhaps even less in
transduced skeletal muscle fibers using AAV-2 vectors)
(Duan et al. 1998a; Nakai et al. 1999, 2001, 2003a, b;
Schnepp et al. 2005; Yue and Duan 2003).
Gene therapy for pulmonary diseases
The first and still most extensive clinical experience with
administration of AAV vectors comes from the treat-
ment of cystic fibrosis (Flotte 2005). These investigations
provided much of the safety profile of the vector and
experience in human application, which paved the
way for subsequent administration to other tissues, as
573
required for other disorders such as hemophilia or
Parkinson’s disease.
Cystic fibrosis
Cystic fibrosis (CF) represents the most common
potentially lethal genetic disease in the Caucasian
population and was one of the first diseases targeted by
gene transfer. The disorder is caused by mutations in the
cystic fibrosis transmembrane conductance regulator
(CFTR), an epithelial chloride channel that also regu-
lates other epithelial ion channels. The most common
mutations of the CFTR (including the Delta-F508
mutation) affect primarily the function of the lung,
where changes in the surface airway fluid and mucous
lead to chronic infection with opportunistic bacteria
such as Pseudomonas aeruginosa and progressive loss
of lung function (Driskell and Engelhardt 2003; Flotte
2005). Changes in viscosity and ionic properties of the
mucous layer lead to reduced clearance and killing of
bacteria. Although CFTR knock out mice are available,
these develop intestinal defects rather than infections of
the lung and are therefore not considered a suitable
model for preclinical studies for CF. Our knowledge on
gene transfer to the lung is derived from studies in in
vitro systems, mice and non-human primates with
normal lung function, and human subjects with CF.
Encouraging for gene therapy, CFTR is expressed at
very low levels, and a small amount of normal CFTR
mRNA or overexpression in 6–10% of polarized airway
epithelial cells may be sufficient to correct the defect in
chloride transport (Chu et al. 1992; Driskell and
Engelhardt 2003; Johnson et al. 1992; Trapnell et al.
1991). On the other hand, CFTR expression is highly
regulated in the lung, and ciliated epithelial cells express
low amounts, while submucosal glands and certain non-
ciliated cells express high levels. It may not be possible to
target submucosal glands by administration of vector
from the airway lumen. Additionally, the mucous layer
in CF patients may impede the delivery of vector par-
ticles to target cells.
Because natural infection with AAV likely occurs in
the respiratory tract, it was necessary to investigate the
possibility of vector rescue upon infection with wild-type
AAV and adenovirus. Studies in non-human primates
showed that this could result in low-level and short-lived
shedding of vector particles (Afione et al. 1996). Fur-
thermore, it was shown that AAV vector delivered to the
lung was predominantly present in episomal forms
(Kearns et al. 1996). Because of the large size of the
CFTR coding sequence (>4.4 kb) and the limited AAV
packaging capacity, expression of early constructs was
driven by promoter elements in the left-hand ITR
(Flotte et al. 1993). Clinical phase I and phase II studies
were based on vector administration to the nasal, sinus,
and bronchial epithelium (Table 1) (Flotte 2005; Flotte
et al. 2003; Wagner et al. 1998a, b, 1999, 2002). Bron-
choscopic and aerosol delivery techniques were used to
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Fig. 2 AAV vector trafficking. AAV-2 binds cell surface heparan
sulfate proteoglycan (HSPG) and is endocytosed via three potential
co-receptors (fgf1r, integrin, and c-met receptors). Endocytosis of
AAV-2 is dependent on dynamin, rac1, and PI kinase activities.
Vector particles escape early and late endosomal compartments
using an inherent phospholipase A2 activity and translocate to the
target the bronchial epithelium. These studies were car-
ried out with AAV serotype 2. Compared to placebo
controls, it was demonstrated that AAV-CFTR gene
transfer resulted in partial correction of the electro-
physiological defect and hyper-inflammatory response
in the maxillary sinus. Aerosol delivery of the vector
improved inflammatory mediators and pulmonary
function (Table 2). Such improvements were observed
during the first 3 months after gene transfer.
In order to increase levels of transgene expression, it
is highly desirable to establish methods that allow
expression from a strong promoter. This requires an
increase in the packaging limit of AAV. In the mid-
1990s, it was established that AAV vector molecules
concatemerize. It became clear that intermolecular
recombination leads to the formation of circles, and that
intermolecular recombination results in head-to-head
and head-to-tail concatemers. Therefore, one can split
the coding sequence into two and package a promoter
construct carrying the 5¢ portion of the cDNA into one
vector, and a construct with the 3¢ portion and a poly-
adenylation signal into a second vector (Nakai et al.
2000b; Sun et al. 2000; Yan et al. 2000). Upon co-
injection of the two vectors, head-to-tail concatemers
lead to transgene expression, provided that appropriate
splice donor and acceptor sites were included in the two
constructs (‘‘split intron’’ approach, Fig. 3b). Appar-
nucleus in a microtubule-dependent fashion. A significant portion
of input vector may be degraded by the proteasome or lysosomal
proteases. While it is still unclear whether intact AAV vectors enter
the nucleus, upon uncoating of the viral genome, second-strand
synthesis provides a transcriptionally active template leading to
expression of the delivered transgene
ently, transcription can proceed through ITR sequences
that remain after the recombination event. Alternatively,
one can produce one vector with a minimal promoter
and the intact coding sequence and provide a second
vector with a strong enhancer (Duan et al. 2000a). A
third strategy relies on homologous recombination be-
tween sequences incorporated into 5¢ and 3¢ sites of the
vector, respectively (Fig. 3c) (Duan et al. 2001). This has
been shown to be quite effective in gene transfer to lung
epithelia (Halbert et al. 2002).
In vitro studies with airway epithelial cell cultures
have demonstrated that AAV-2 is limited in its ability to
transduce these cells despite efficient viral entry on the
apical surface of these polarized cells. The vector particle
mostly fails to translocate to the nucleus and remains in
the cytoplasm (Duan et al. 1998b). This defect in traf-
ficking does not occur if the vector is applied to the
basolateral surface, which is not accessible during gene
transfer in vivo. Proteasomal degradation limits transfer
of the vector from endosomes to the nucleus, which can
be blocked by certain antibiotics and other proteasome
inhibitors (Duan et al. 2000b). On the other hand,
ubiquitination seems required for transduction from the
apical surface. Interestingly, other serotypes such as
AAV-5 transduce from the apical surface with substan-
tially greater efficiency (Zabner et al. 2000). AAV-1, -5,
and -6 show improved transduction efficiency compared
Table 1 Summary of clinical trials for treatment of human diseases by AAV gene transfer

Disease Transgene Serotype Route of administration Target cell Clinical trials

a1-Antitrypsin deficiency a1-Antitrypsin AAV-2, AAV-1 Intramuscular Skeletal muscle fibers Phase I/II
Alzheimer’s disease Nerve growth factor AAV-2 Intracranial or ex vivo Neurons Phase I/II
Canavan’s disease Aspartoacylase AAV-2 Intracranial Neurons Phase I
Cystic fibrosis CFTR AAV-2 Nasal, sinus, and Lung epithelial cells Phase I, phase II
bronchial epithelium
Hemophilia B Factor IX AAV-2 Intramuscular Skeletal muscle fibers Phase I/II
Hemophilia B Factor IX AAV-2 Hepatic artery Hepatocytes Phase I/II
Leber congenital RPE65 AAV-2 Subretinal space Photoreceptors Phase I/II approved
amaurosis (blindness)
Parkinson’s disease Aromatic L-amino acid AAV-2 Intracranial Neurons Phase I
decarboxylase
Muscular dystrophy Micro-dystrophin AAV-1/2 hybrid Intramuscular Skeletal muscle fibers Phase I initiated
Parkinson’s disease Glutamic acid decarboxylase AAV-2 Intracranial Neurons Phase I

Table 2 Summary of published results from clinical trials for treatment of human diseases by AAV gene transfer

Disease Clinical outcome Limitations Potential future directions Reference

[# patients treated]

Cystic fibrosis [200] –Safe gene transfer to nasal sinus
and bronchio-epithelium
–Partial correction of electrophysiologic
defect and hyperinflammatory responses
–Improved pulmonary function
Hemophilia B –Safe gene transfer to skeletal muscle
(muscle-directed) [8] –Evidence for transduction of
muscle in all patients
Hemophilia B –Safe vector infusion to hepatic artery
(hepatic) [7] by angiographic procedure
–Therapeutic levels (>10%) at
highest vector dose
–Limited duration of biological effect
–Failure to stably transduce
progenitor cells
–Sub-therapeutic F.IX levels
–Dose escalation limited by vector
dose per injection site and total
number of injections
–Potential risk of inhibitor formation as
evidenced by preclinical studies
–Transient F.IX expression (<8 weeks)
–Transient transaminitis in 2 subjects due
to T cell response to AAV capsid
–Lack of gene transfer to patients
with high neutralizing antibody titer
–Alternative serotypes with Wagner et al. (1998b, 1999),
improved transduction in lung Moss et al. (2004)
–Re-administration protocols
–Development of more efficient
integrating vector system
–Vascular delivery for widespread Kay et al. (2000),
muscle gene transfer Manno et al. (2003)
–Transient immune suppression
–Alternative serotypes
or hybrid vectors
–Transient immune suppression Manno et al. (2006)
–Alternative serotypes
575
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Fig. 3 Strategies to overcome the size limitation of the recombi-
nant AAV genome. a Standard vector design. b Dual vector that
yields expression after recombination (head-to-tail end joining) and
cis-splicing. c Dual vector that yields expression after homologous
recombination. d Dual vector expressing two chains of a protein
separately, which in certain cases can assemble intracellularly into a
functional protein. e Dual vector expressing two chains of a protein
separately, which can form a single chain functional protein after
to AAV-2 (Halbert et al. 2001; Zabner et al. 2000). For
example, the primary receptor for AAV-5 is more
abundant on the apical surface of airway cells than the
receptor for AAV-2.
Once the vector particle has uncoated, the single-
stranded genome needs to become double-stranded to
achieve transgene expression. The nuclear protein
intracellular protein splicing. This is accomplished by addition of
split intein sequences (a segment of a protein that is able to excise
itself and rejoin the remaining ends with a peptide bond) to the C or
N terminus of the two chains. f Single vector expressing a pre-trans-
splicing molecule (PTM). This strategy relies on the presence of
transcript in order to obtain an mRNA encoding a functional
protein after trans-splicing
FKBP52 in a dephosphorylated form binds the D se-
quence of the AAV-2 ITR and thereby prevents second-
strand synthesis (Mah et al. 1998; Qing et al. 1997,
2001). FKBP52 is mostly present in phosphorylated
form in skeletal muscle, but is mostly dephosphorylated
in lung and other tissues (Qing et al. 1998). Therefore,
one should be able to improve the efficacy by adminis-

tration of tyrosine phosphatase inhibitors or by co-
administration of a second vector that expresses a
phosphatase such as T cell-derived protein tyrosine
phosphatase (Zhong et al. 2004). This strategy has al-
ready been shown to be successful in hepatic gene
transfer with AAV. Expression of the phosphatase was
from a self-complementary AAV vector. Such vectors
are engineered to contain a double-stranded genome by
manipulation of the D sequence of the ITR and by
containing an expression cassette that is half the size of a
traditional AAV genome and that is present as a repeat,
with the second copy representing a reverse comple-
mentary form of the first copy (McCarty et al. 2001).
As pointed out above, a therapeutic effect of AAV
gene transfer to the lung is likely to be transient, which
may be due to a lack of transduction of stem or pro-
genitor cells and/or integration of vector genomes. In
this case, transduced epithelial cells that turn over are
not replaced with cells that carry the vector genome.
Strategies may be devised that increase integration
(preferably in a targeted way). Alternatively, use of
alternate serotypes results in successful re-administra-
tion in animals studies, and some efficiency of re-
administration of the same serotype has been described
for AAV-6 in the lung (Halbert et al. 2001).
Therapy for a1-antitrypsin deficiency
Functioning as a protector from proteases such as neu-
trophil elastase, the systemic protein a1-antitrypsin is
normally synthesized in the liver. Deficiency in a1-anti-
trypsin, typically caused by a missense mutation that
abrogates function but does not eliminate systemic
expression, causes panacinar emphysema of the lung and
cirrhosis of the liver. In particular the resulting increase
in neutrophil elastase activity causes recurrent digestion
of the extracellular matrix in the lung and life-threat-
ening disease (Driskell and Engelhardt 2003; Flotte
2005). Presence of ‘‘non-functional’’ a1-antitrypsin pro-
tein in the circulation of patients should substantially
reduce the likelihood of an immune response to the
functional therapeutic gene product. A certain threshold
level of systemic expression of functional a1-antitrypsin
has to be achieved to prevent the disease (0.6–0.8 mg/
ml). Feasibility of AAV-2 gene transfer to skeletal
muscle or liver has been demonstrated in mouse and
large animal studies, where high and sustained systemic
levels of expression were obtained (Flotte 2005; Song
et al. 1998, 2004). More recently, a report describing the
administration of AAV-2 a1-antitrypsin to the pleural
space has been suggested (De et al. 2006). While the liver
offers a distinct vector dose advantage, muscle was
chosen for an initial clinical trial because of the non-
invasive nature and ease of administration and limited
bio-distribution of vector sequences (Flotte 2005; Flotte
et al. 2004). After demonstration of a favorable safety
profile, the trial is continued with the modification of use
of AAV serotype 1 capsid (Table 1). Xiao et al. (1999)
577
have initially described that the AAV-1 capsid yields 1
log increase in transgene expression compared to AAV-2
in skeletal muscle, and subsequent work by others
showed that the increase in efficacy may be even greater
with other transgenes (Arruda et al. 2004a; Chao et al.
2000). In contrast to AAV-2, AAV-1 efficiently trans-
duces fast-twitch muscle fibers. Transfer of a functional
a1-antitrypsin gene to skeletal muscle using AAV-1 may
achieve the desired therapeutic target. Experience with
muscle-directed AAV-1 gene transfer in this trial could
also yield valuable information for treatment of mus-
cular dystrophy.
Gene therapy for hemophilia
Hemophilia is the X-linked bleeding disorder caused by
mutations of coagulation factor VIII (hemophilia A) or
IX (hemophilia B) and occurs with a frequency of 1 in
5,000 male births worldwide. Hemophilia A is the more
common form (approximately two-thirds of cases) of the
phenotypically identical bleeding diatheses. Severe dis-
ease generally correlates with protein levels below 1% of
normal, which typically results in frequent spontaneous
bleeds into joints and other soft tissues. Bleeds into
critical closed spaces (e.g., intracranial or retroperito-
neal) can be fatal. Severity of the disease directly
correlates with factor levels; levels of 1–2% are likely
sufficient to prevent severe disease and levels >5% are
associated with only mild disease. Functional coagula-
tion factors can be expressed in various cell and tissue
types upon gene transfer. Small and large animal models
of hemophilia facilitate preclinical testing of novel
treatment strategies. Additionally, clinical end points
required to assess efficacy are easily measurable and
correlate well with clinical outcome.
Gene therapy for hemophilia A
Expression of factor VIII (F.VIII, the co-factor of the
serine protease factor IX, F.IX) from an AAV is made
difficult because of the large size of the F.VIII cDNA.
In fact, the full-length cDNA cannot be incorporated
into an AAV vector. However, B domain-deleted
F.VIII, which is functional and used as a recombinant
protein in conventional therapy, can be expressed. This
requires use of a small promoter and short sequences
for other regulatory elements (intron, polyA signal,
etc.). F.VIII is normally synthesized in the liver by
hepatocytes and endothelial cells. AAV-2, when intro-
duced into the liver by injection into the portal vein,
can transduce 5% of hepatocytes and provides vector
dose-dependent levels of expression. Given these limi-
tations, only F.VIII levels at the lower end of the
therapeutic range were achieved in hemophilia A mice
and dogs (Sarkar et al. 2003; Scallan et al. 2003a).
Nonetheless, sustained levels (>1 year) of 2–4% of
normal were documented in the canine model, which
578
correlated with a lack of spontaneous bleeding. To
achieve this level of correction, large vector doses were
required (6·1012 vector genomes [vg]/kg or 2.7·1013 vg/
kg). Much higher levels of gene transfer to murine liver
were obtained with vector packaged into the capsid of
serotype 8 (originally isolated from a non-human pri-
mate) (Sarkar et al. 2004). In order to circumvent the
packaging limit of AAV, several approaches have been
pursued. In the case of F.VIII, the coding regions
for the heavy and light chains of the F.VIII protein
have been expressed from separate vectors, which
are administered simultaneously (Burton et al. 1999;
Scallan et al. 2003b). The two chains are capable of
assembling in the cell prior to secretion of a functional
F.VIII molecule (Fig. 3d) (Mah et al. 2003). This led to
complete correction of murine hemophilia A, which can
be done with relatively low vector doses, if AAV-8
vectors are used (Sarkar et al. 2004). However, the
amount of F.VIII heavy chains made in vivo was 25- to
100-fold less than that of the light chains by this sys-
tem, even though both vectors used the same liver-
specific promoter. The differential expression of light
chains appeared unrelated to transduction efficiency or
to mRNA transcription, but instead was most likely
due to relatively lower heavy chain translation or pro-
cessing or stability (intracellular half-life) (Scallan et al.
2003b). For reasons that are not fully understood, the
heavy/light chain dual AAV-8 vector protocol has been
much less efficient in hemophilia A dogs than in mice
(unpublished observations by Sarkar and Kazazian).
The high efficiency of AAV-8 transduction of murine
hepatocytes has in part been attributed to substantially
more rapid uncoating compared to AAV-2 (Thomas
et al. 2004). As a consequence, the kinetics of transgene
expression is different between the two serotypes. AAV-
2 directs only low levels of transgene expression early
after gene transfer that gradually increase to a stable
plateau within 1 month. In the case of AAV-8,
high levels of expression are observed early, which
decline over several weeks to reach a plateau that (in
mice) is >1 log higher than with a comparable dose of
AAV-2 vector (Davidoff et al. 2005a).
In a second dual-vector approach, F.VIII transgene
was carried in two AAV vectors, each harboring the 5¢
or 3¢ half of the cDNA, with intronic sequences and
intron splice donor/acceptors that permit splicing of the
RNAs to reconstitute a complete F.VIII mRNA after
intermolecular recombination between the two genomes
(Fig. 3b) (Chao et al. 2002). However, the efficiency
was low, and expression levels were lower than from
a single F.VIII vector strategy. It is conceivable that
optimization of the positioning of splice donor and
acceptor sites may increase efficacy. Trans-splicing is
also under investigation for AAV-mediated treatment of
hemophilia (and muscular dystrophy, see below). In this
case, a single vector expressing a pre-trans-splicing
molecule (PTM) is administered to the liver (Chao et al.
2003). If a F.VIII transcript is present in hepatocytes
and base pairing between the non-functional transcript
and the PTM can be engineered to occur before the
mutation, trans-splicing would result in an mRNA
encoding a functional F.VIII protein (Fig. 3e), albeit
that trans-splicing is less efficient than cis-splicing.
Muscle-directed gene transfer for hemophilia B
Because of the smaller size of coding sequence (1.4 kb),
AAV vectors can easily accommodate a F.IX expression
cassette featuring a strong promoter element. While
F.IX is normally synthesized by hepatocytes, muscle
fibers are capable of producing biologically active F.IX
that is similar to hepatocyte-derived protein with regard
to post-translational modifications (Arruda et al. 2001a).
A simple intramuscular (IM) injection of AAV vector
can direct sustained therapeutic levels of FIX in mice
(Herzog et al. 1997). Levels of expression are vector
dose-dependent and reach a stable plateau 6–8 weeks
after vector administration. In the late 1990s, scale-up
studies in hemophilia B dogs were carried out, in which
AAV serotype 2 was administered percutaneous at
multiple IM sites at a single time point (Herzog et al.
1999). Sustained expression and shortening of in vitro
coagulation times were observed for as long as animals
were followed (>4 years for several dogs). However,
large vector doses (1013 vg/kg) were required to achi-
eve therapeutic levels of expression (1–2% of normal).
For safety reasons and because of the simplicity of the
procedure, the IM route was the protocol of choice for a
first clinical trial that involved parental administration
of AAV vector.
In a phase I dose escalation study, AAV-2 vector
expressing F.IX from the strong viral CMV enhancer/
promoter was introduced into the skeletal muscle of the
upper and lower extremities of eight hemophilia patients
by IM injection (Table 1) (Kay et al. 2000; Manno et al.
2003). Muscle biopsies taken 2–10 months post-injection
demonstrated gene transfer and/or transgene expression
by DNA analyses and immunohistochemistry in all
subjects. Preexisting antibodies to AAV did not block
gene transfer. Vector doses were up to 1.8·1012 vg/kg
in three dose cohorts. Important safety parameters
included risks of antibody formation to F.IX, germline
transmission of vector sequences, and insertional muta-
genesis. This clinical study demonstrated the safety of
IM injection of AAV-F.IX, in that none of the patients
developed antibodies to FIX, and that none showed
evidence of vector sequence in the semen (Arruda et al.
2001b; Manno et al. 2003). In up to 6 years of follow-up,
none has shown evidence of tumor formation (High
2004). However, systemic F.IX levels were generally less
than 1%, and dose escalation to levels that proved
therapeutic in animals is likely not feasible because of
the large number of injection sites that would
be required. The dose per site is limited by saturation
with vector particles, decreased specific activity of F.IX
overexpressed in muscle fibers, and an increased risk of
immune responses at high local antigen doses (Herzog

et al. 2002). Nonetheless, the clinical study demonstrated
that injection of AAV-2 into skeletal muscle was safe at
the doses tested and could result in long-term expression
of F.IX (Table 2). Moreover, the general characteristics
of transduction of human muscle such as molecular
forms of the vector genome were similar to those
observed in animal models (Manno et al. 2003). AAV
serotype 2 preferentially transduces slow-twitch myofi-
bers (which express a particular isoform of myosin and
HSPG, the primary receptor for AAV-2). This pattern
was also seen in human muscle. The safety and efficacy
data established in the first trial formed the basis for a
second trial in which AAV-FIX is administered sys-
temically to target the liver.
Subsequent preclinical studies in animals have shown
substantial improvement of F.IX gene transfer to skel-
etal muscle can be achieved by switching to a different
serotype that is not limited to transduction of slow-
twitch fibers (such as AAV-1, -6, -7, -8, or -9). Use of
these capsids can increase F.IX gene transfer and
expression by >1 log (Chao et al. 2000; Gao et al. 2002,
2004; Xiao et al. 1999). However, the safety of using
these vectors in the treatment of hemophilia is unclear.
Increased local levels of F.IX expression raised the
likelihood of antibody formation to F.IX, unless very
high levels of expression (>10%) are achieved. This
may represent a high dose tolerance phenomenon
(Arruda et al. 2004a; Chao et al. 2001). Alternatively,
AAV-2 vector can be delivered to skeletal leg muscle
through a vascular route by means of isolated limb
perfusion or retrograde infusion via the superficial
saphenous vein (Arruda et al. 2004b, 2005). The first
method requires administration of drugs that cause
vasodilatation and increased vascular permeability,
while the second technique relies on pressure through
injection of a high volume in a short period of time for
efficient translocation of vector from the blood vessel
into muscle tissue. Limb perfusion demonstrated long-
term (>3 years), robust F.IX expression (circulating
levels of 4–14%) by muscle-directed gene transfer,
resulting in nearly complete correction of the bleeding
disorder in hemophilic dogs (Arruda et al. 2005).
Transient immune suppression was required to block
anti-F.IX formation (see below). Additionally, incor-
poration of novel variants of F.IX with increased spe-
cific activity or decreased binding to the extracellular
matrix of skeletal muscle provides additional tools for
increased efficacy (Schuettrumpf et al. 2005).
Liver-directed gene transfer for hemophilia B
Sustained high levels of F.IX have been demonstrated in
mice, dogs, and non-human primates after injection of
AAV-2 into the portal vein or hepatic artery (Mount
et al. 2002; Nathwani et al. 2002; Snyder et al. 1997,
1999; Wang et al. 1999, 2000b). This led to complete
correction of murine hemophilia B and substantial cor-
rection of canine hemophilia B after portal vein infusion
579
of 1·1012 vg/kg of an AAV-2 vector with an apoli-
poprotein A/a1-antitrypsin enhancer/promoter combi-
nation (Mount et al. 2002). Expressing canine F.IX
(cF.IX) in the hemophilic dogs showed >3 years
circulating levels of F.IX (>5% or normal; Herzog,
Nichols, Lothrop, and High, unpublished data).
A liver-directed phase I dose escalation study was
initiated in 2001 (Table 1) (Manno et al. 2006). Seven
patients have been studied at doses ranging from 8·1010
to 2·1012 vg/kg. All subjects had severe hemophilia B.
Vector was administered to the hepatic artery using an
interventional radiology technique. The four subjects
treated in the first two dose cohorts not only showed no
vector-related toxicity but also failed to achieve F.IX
levels above baseline. In all subjects tested so far, DNA
extracted from the semen has been transiently positive
for vector sequences, with all patients negative at the end
of the trial. None of the seven patients have developed
antibodies to F.IX. The patient who was the first to
receive a dose of 2·1012 vg/kg showed circulating F.IX
levels of >10%, which persisted for 4 weeks and then
gradually fell to the baseline of <1% (Table 2). Con-
comitant with the fall in F.IX level was a rise in trans-
aminase levels, first noted 4 weeks after injection,
peaking at 5 weeks, and then returning to normal. This
patient had low baseline anti-AAV antibody titers (1:2).
The transient elevation in transaminases has been
interpreted as an immune-mediated event upon sub-
sequent analysis, likely caused by memory CD8+ T cell
responses directed against AAV capsid (Manno et al.
2006). Capsid-specific T cells may have been activated
during the course of an earlier natural infection with the
wild-type virus in the presence of a helper virus. A T cell
response to AAV capsid was demonstrated in a subse-
quently treated subject, who also had low antibodies to
AAV prior to gene transfer but received a lower vector
dose. High-titer preexisting neutralizing antibodies to
AAV appeared to prevent efficient gene transfer.
It is important for future clinical success to under-
stand that humans, as opposed to most animal models
currently available for preclinical studies, are natural
hosts for AAV. In contrast to vectors, natural infection
may occur in the presence of a helper virus, which is
likely to augment T cell responses to AAV. The impact
of preexisting/memory T cells responses to AAV thus
deserves more extensive investigation. Future studies
may also address whether T cells responses to AAV
capsid can be blocked by optimized protocols for tran-
sient immune suppression.
Some AAV serotypes (such as AAV-6 and -8) show
similar efficacy in hepatic gene transfer for injection
from a peripheral vein as for portal vein administration
(Grimm et al. 2003; Nakai et al. 2005a). As discussed
above, AAV-8 is an attractive candidate for hepatic gene
transfer applications since 10- to 100-fold improved
transduction efficiency has been achieved in mouse
liver. Studies performed by Davidoff et al. (2005a) have
shown that liver-targeted administration of AAV-8
and AAV-5 vectors established therapeutic levels of
580
transgene expression in non-human primates. Recently,
Wang et al. (2005a) demonstrated the safety and imp-
roved efficacy of AAV-2/8 vector in large-animal models
for liver-directed gene therapy. In an AAV2-pretreated
hemophilia B dog, canine FIX expression increased from
<1 to 16% of normal levels when AAV-8 vector carrying
the same gene was subsequently administrated. This
high-level F.IX expression has lasted for more than
2 years. No significant liver toxicity or cFIX-specific
antibodies have been detected. Thus, serotype-specific
differences in transduction biology and appreciation of
preexisting immunity will likely influence the selection
of the AAV serotype for future clinical trials. It should
be pointed out that cross-reactivity among alternate
serotype on the T cell level, which may result from
natural infection with AAV, is unclear at this point.
Immunology of AAV-F.IX gene transfer
Depending on the underlying F.IX mutation and other
genetic factors, the recipient of therapy may not be
tolerant to wild-type F.IX (Sabatino et al. 2004). As a
result, inhibitory antibodies to F.IX may form (termed
‘‘inhibitors’’) (Herzog and Dobrzynski 2004). This may
be of even greater concern in the treatment of hemo-
philia A with F.VIII gene therapy since 20–30% of
hemophilia A patients form inhibitors after conven-
tional protein-based therapy. Synthesis of these anti-
bodies by B cells is dependent on the activation of T
help (i.e., CD4+ T cells). If the recipient of AAV-F.IX
gene transfer lacks tolerance to the F.IX transgene
product, intramuscular administration of the vector
typically results in a local immune response, which
causes activation of CD4+ T and B cells in the
draining lymph nodes of the transduced skeletal muscle
(Wang et al. 2005b). Activation of CD8+ T cells
(cytotoxic T lymphocytes) to F.IX is comparatively
inefficient. In a series of studies in hemophilia B mice
and dogs, transient immune suppression with the drug
cyclophosphamide was shown to be effective in pre-
venting the inhibitor response, thereby leading to sus-
tained expression (Fields et al. 2001). The drug, which
is a DNA alkylating agent toxic to dividing cells such
as activated lymophocytes, is given every 2 weeks
starting on the day of vector administration and ending
at week 6 (Fields et al. 2001; Herzog et al. 2001).
Interestingly, experiments in mice and dogs have yiel-
ded sustained hepatic F.IX expression in animal models
that consistently showed inhibitor formation in the
context of muscle-directed gene transfer (Mingozzi
et al. 2003; Mount et al. 2002; Nathwani et al. 2001).
A detailed study in mice demonstrated that the hepatic
route can induce antigen-specific immune tolerance
to the F.IX transgene product (Mingozzi et al. 2003).
Tolerance was obtained in adult mice and was main-
tained even after administration of F.IX protein
formulated in adjuvant. Tolerance induction was
associated with lack of B and T helper cell responses to
F.IX and activation of regulatory CD4+ T cells
capable of suppressing antibody formation to F.IX
after adoptive transfer (Mingozzi et al. 2003; Wang
et al. 2005b). Activation of regulatory T cells also
suppressed inflammatory cellular immune responses
against hepatocytes transduced with F.IX expressing
vectors (Dobrzynski et al. 2006). Analogous studies
with ovalbumin transgene in T cell receptor transgenic
mice provided direct evidence for CD4+ T cell toler-
ance (T cell anergy and deletion) (Dobrzynski and
Herzog 2005; Dobrzynski et al. 2004). Similar results
showed that antibodies to other transgene products can
be avoided by restricting the expression to hepatocytes,
which promotes immune tolerance (Franco et al. 2005;
Herzog 2005; Ziegler et al. 2004). These observations
have implications for treatment of other protein defi-
ciencies, in particular lysosomal storage disorders (see
below). As a result of the assessment of the risk of
inhibitor formation in animal models of hemophilia B,
the vector dose per site was restricted in the muscle-
directed clinical trial and only subjects with F.IX mis-
sense mutations were enrolled. This restriction was not
applied to the clinical study on hepatic gene transfer
(Manno et al. 2003, 2006).
AAV-mediated gene transfer for lysosomal storage
disorders
Lysosomal storage disorders (LSDs) constitute a diverse
group of recessive genetic diseases caused by deficiencies
of cellular acid hydrolases that normally catalyze the
breakdown of glycoproteins, glycolipids, and other
macromolecules or from defects in transporter proteins
leading to pathogenic accumulation of these substances
in lysosomes (Cheng and Smith 2003; Ellinwood et al.
2004). Current treatment options include bone marrow
transplantation and enzyme replacement therapy. LSDs
can affect a limited set of cell types found in particular
tissues or a large number of cells and tissues, depending
on the defective gene. Frequently, the disease is associ-
ated with neurodegeneration in the CNS or demyeliza-
tion affecting the entire nervous system.
These are rare but devastating diseases. Progression
of the disease is often lethal during infancy and treat-
ment is suboptimal, thus making gene therapy highly
desirable. Despite accumulation of compounds in the
lysosomes of cells in multiple tissues, correction of such
a disease may be achieved by gene transfer to one or few
tissues or even a limited number of cells within a tissue
because of cross-correction. If one can achieve adequate
levels of systemic expression of the missing enzyme by
gene transfer, this may be sufficient to achieve uptake of
the enzyme by cells in other tissues (e.g., through the
mannose-6-phosphate receptor), thereby providing a
therapeutic effect. Preclinical testing of novel therapies is
facilitated by the availability of mouse models for these
diseases and in some cases also large animal models
(dogs and cats) (Ellinwood et al. 2004).

Mucopolysaccaridosis type VII
Treatment for mucopolysaccaridosis type VII (MPS VII,
Sly disease) is extraordinarily difficult because it affects
multiple organs. This disorder is caused by deficiency in
b-glucuronidase (GUSB) and severely interferes with
bone and joint development, eye, brain, and heart
function, and other organs. Following the premise that
treatment should be most effective in early stages of life,
neonatal MPS VII mice were tested for efficacy of AAV-
mediated gene transfer (Daly et al. 1999). Intravenous
injection of vector resulted in persistent enzymatic
activity in many tissues, including brain, liver, lung,
heart, kidney, and spleen. Gene transfer to multiple
tissues was achieved, including brain, which may be due
to a not completely intact blood–brain barrier in neo-
natal animals. It is unclear whether such widespread
transduction can be achieved in older animals or hu-
mans. In an effort to achieve systemic protein delivery,
the hypothesis was developed that the liver can serve as
an enzyme depot by overproduction following hepatic
gene transfer, and vascular dissemination of the gene
product would cause a reduction in lysosomal storage in
other tissues. To test this, hepatic gene transfer with an
AAV-GUSB vector was carried out in adult MPS VII
mice, resulting in 500% of normal enzyme levels in the
liver and substantial reduction of storage lesions in
spleen, kidney, heart, lung, and brain (Sferra et al. 2004).
Some vectors had also disseminated to the brain. Be-
cause the study did not utilize a hepatocyte-specific
promoter, it was unclear if correction in neuronal, glial,
and perivascular cells was achieved by GUSB expressed
in the brain or by cross-correction by liver-derived en-
zyme. However, data from a retroviral vector system
also support the interpretation that at least some level of
correction in the brain can be achieved by systemic
GUSB expression (Ponder et al. 2002). The possibility of
reducing storage by direct administration of AAV vector
to the brain will be discussed in Targeting CNS for
treatment of lysosomal storage disorders.
Glycogen-storage disease type II
Deficiency in acid a-glucosidase (GAA) results in gly-
cogen-storage disease type II (GSD-II, Pompe disease),
which is characterized by myopathy in skeletal, heart,
and diaphragm muscles. Respiratory or cardiac failure
typically occurs within 2 years of age. Gene transfer to
skeletal and heart muscles has been demonstrated to
improve the function of transduced tissues. While
achieving local correction, using muscle as a depot for
the enzyme has had very limited success (Fraites et al.
2002; Sun et al. 2005b). Use of a muscle-specific pro-
moter reduced immune responses to transduced tissue
(Sun et al. 2005b). While hepatic gene transfer led to
significant correction of diaphragm and heart function,
reduction of storage in skeletal muscle by cross-correc-
tion has been difficult in Pompe disease (Cresawn et al.
581
2005). Recent studies demonstrate that it is possible to
achieve correction in skeletal muscle (Franco et al. 2005;
Sun et al. 2005a). A strong hepatocyte-specific promoter
was used to express GAA from an AAV-2 genome
packaged into AAV-8 capsid, which, as outlined above,
directs high levels of gene transfer to the liver. Equally
important, correction was obtained in immune compe-
tent GAA-deficient mice (Franco et al. 2005). Similar to
observations in hemophilia gene therapy (vide infra),
restricting expression to hepatocytes was a successful
maneuver to avoid antibody as well as CD4+ and CD8+
T cell responses to the transgene product. An alternative
approach to prevent fatal cardiomyopathy and muscular
dystrophy in GSD II is systemic administration of a
vector during the neonatal period, which was success-
fully done in GSD II mice using an AAV-1 vector (Mah
et al. 2005).
Fabry disease
Fabry disease is an X-linked defect caused by deficiency
in a-galactosidase A. Increased storage in lysosomes of
vascular endothelial cells causes renal, cardiovascular,
and cerebrovascular disease. Data from treatment of
mice with Fabry disease illustrate that hepatic gene
transfer has the capacity to induce immune tolerance to
lysosomal storage enzymes, similar to induction of tol-
erance to F.IX in hemophilia B (Ziegler et al. 2004). In
the murine model, animals with persistent systemic gene
expression failed to form antibodies to a-galactosidase A
after administration of the protein in adjuvant. A certain
level of expression was required to obtain tolerance.
Gene therapy for retinal diseases
The mammalian retina is organized in a laminar struc-
ture composed of three layers of neuronal cells, with
photoreceptor cells being located in the deepest retinal
layer. The outer segments of the photoreceptors consist
of membranous disks containing rhodopsin or cone
opsin molecules for light capture. Each day, approxi-
mately 10% of each outer segment is shed and regen-
erated. A monolayer of post-mitotic cells called retinal
pigmented epithelium (RPE) is located behind the ret-
ina. The RPE is in close contact with the photoreceptor
layer, to which it is connected through an extracellular
matrix. For transduction of photoreceptors or RPE
cells, a small volume of highly concentrated vector is
typically injected into this subretinal space (Dinculescu
et al. 2005). The RPE is critically linked to the function
of photoreceptors. RPE cells take up vitamin A, provide
nutrients, growth hormones, and oxygen to photore-
ceptors, and phagocytose mature disks shed from the
outer segments of photoreceptors. Because photorecep-
tors and RPE are functionally so tightly linked, muta-
tions in genes expressed by one cell type will inadvertently
affect the other. Consequently, RPE malfunction leads to
degeneration of photoreceptors.
582
Retinal diseases comprise a large number of genetic
defects, with 150 genetic loci mapped in the human
genome (Dinculescu et al. 2005). Several murine and
large animal models are available to test gene transfer
strategies for treatment of retinal diseases. Since pho-
toreceptors are post-mitotic cells that normally cannot
be replaced, treatment has to be done as early as possible
in order to prevent retinal degeneration.
Serotypes of AAV differ in the efficiency with which
they transduce retinal cells. Differences in their tropism
can also be exploited for targeting different cell types.
For example, AAV-5 was shown to transduce >2 logs
more photoreceptor and RPE cells than AAV-2 in
mouse retina (although tropism of AAV-5 was more
restricted to photoreceptors in the retina of non-human
primates) (Lotery et al. 2003; Yang et al. 2002). AAV-1
predominantly transduces RPE cells, again with higher
efficiency than AAV-2, and AAV-4 exclusively targets
the RPE (Weber et al. 2003; Yang et al. 2002). Specificity
of transgene expression can be further improved by
use of tightly regulated promoters. While promoters
from viral and housekeeping genes may be suitable if
expression in photoreceptor and RPE cells is desired, cell
type-specific expression has been documented for pho-
toreceptors with a proximal murine rod opsin promoter
and for RPE cells with promoter elements isolated from
VMD2 or RPE65 genes (Dinculescu et al. 2005). Vari-
ation in the injection method can be applied as well.
Retinal ganglion cells can be targeted by intravitrial
injection.
Leber congenital amaurosis
In the following, we will provide several examples of
retinal diseases that were successfully treated in animal
models by AAV gene transfer. Humans with the auto-
somal recessive disorder Leber congenital amaurosis
(LCA) are born with vision deficits, and the disease
progresses to blindness by age 10. A proportion of cases
(10–15%) is caused by mutations in the RPE65 gene.
RPE65 plays a critical role in the retinoid cycle, in which
absorption of light by rhodopsin in the photoreceptor
results in isomerization and reduction of retinal, which is
transported to and ultimately recycled in the RPE. There
is a large animal model for this form of childhood
blindness (RPE65-deficient Briard dogs). In 2001, it was
shown that subretinal delivery of an AAV-2 vector
expressing functional RPE65 halted retinal regeneration
and restored vision in these animals (Acland et al. 2001;
Narfstrom et al. 2003a). This was demonstrated by his-
tochemistry, electroretinography, and behavioral stud-
ies. Marked improvements in vision were observed as
early as 4 weeks after gene transfer and could be docu-
mented in long-term follow-up studies (Narfstrom et al.
2003b). These results provided the basis for preparation
of a clinical trial for treatment of LCA, which is
scheduled for the near future (Table 1). It should be
pointed out that in other disorders, depending on the
rate of retinal degeneration, treatment may require
earlier intervention than reported in the canine LCA
study. To that end, successful treatment of RPE65
knock out mice was reported for in utero gene transfer
with AAV-1 vector (Dejneka et al. 2004).
Juvenile retinoschisis
Mutations in the RS1 gene cause a degeneration of the
central retina, which manifests in a disease termed
juvenile retinoschisis (RS). Retinoschisin is critical for
maintenance of synaptic integrity of the retina, and
retinal splitting is characteristic for RS. Subretinal
injection of an AAV-5 vector expressing wild-type
human RS1 from the mouse opsin promoter into RS1-
deficient mice at day 15 of age restored visual function,
reduced retinal splitting, and greatly improved structural
integrity of the retina (Min et al. 2005). Improvement of
visual functions could be demonstrated several months
after gene transfer, indicating a long-term therapeutic
effect.
Retinitis pigmentosa
While the above-discussed examples of ocular diseases
reflect protein deficiencies that can be treated by gene
replacement therapy, other disorders may have a dom-
inant effect caused by toxic accumulation of a dysfunc-
tional gene product. In this case, strategies have to be
developed that decrease the expression of an endogenous
gene without affecting the expression of the functional
protein. The term retinitis pigmentosa (RP) describes
a family of genetically heterogeneous diseases, which
result in night blindness and loss of peripheral vision
secondary to loss of rod photoreceptors. Loss of cones
and central vision occurs later during disease progres-
sion. Mutations in the rhodopsin are responsible for the
largest proportion of autosomal dominant RP (ADRP).
A ribozyme-based treatment approach has been devised
to target toxic gain of function mutations (Lewin et al.
1998). Hairpin and hammerhead ribozymes were de-
signed and optimized in vitro to target the mutant
mRNA expressed in a transgenic rat model of ADPR.
Subsequent in vivo studies in these animals showed that
AAV at day 15, 30, or 45 of life delayed the loss of
photoreceptor cells. Improved retinal function and sur-
vival of rods was observed even at 8 months of age
(LaVail et al. 2000).
Neovascular retinal disease
Neovascular retinal disease represents yet another dif-
ferent challenge for treatment. Abnormal formation of
new capillaries from preexisting blood vessels is char-
acteristic for the pathology of several prevalent diseases
of the eye, such as diabetic retinopathy and age-related

macular degeneration, and therefore contributes to the
most common causes of blindness in developed coun-
tries. Current treatments only deal with the symptoms
of the disease rather than with the underlying cause
of neovascularization, are often destructive to existing
tissue, and repeated intervention is typical (Dinculescu
et al. 2005). It is therefore highly desirable to develop a
protocol that suppresses abnormal vessel formation.
Overexpression of vascular endothelial growth factor
(VEGF) or an imbalance between VEGF and its
antagonists (expressed locally in the tissue) is a key
feature of ocular neovascularization. Increasing con-
centrations of VEGF specifically cause proliferation of
endothelial cells (such as vascular endothelial cells). It is
thought that pigment epithelium-derived factor (PEDF)
is an important angiostatic factor that counteracts
VEGF in vivo (Behling et al. 2002). PEDF is highly
expressed in the normal eye to prevent vascularization of
the cornea and the vitreous. Several animal models have
been developed that can be used to test the effect of
angiostatic factors on the treatment of neovascular
retinal disease. These include laser-induced choroidal
neovascularization (CNV), oxygen-induced retinopathy
(OIR), and transgenic mice overexpressing VEGF
(Dinculescu et al. 2005). Potential therapeutic molecules
that can be expressed from an AAV vector in the eye
include PEDF, angiostatin, endostatin, a soluble form of
VEGFR1 receptor, short peptides corresponding to a
portion of VEGF exon 6 (which can compete with
binding of VEGF to VEGR2 receptor), and others
(Auricchio et al. 2002; Bainbridge et al. 2002, 2003; Lai
et al. 2005b; Raisler et al. 2002). In particular, AAV-
mediated transfer of a PEDF cDNA shows promise for
prevention of retinal neovascularization (Auricchio et al.
2002; Mori et al. 2002).
Gene therapy for muscular dystrophy
Duchene muscular dystrophy is a severe X-linked
disorder characterized by inherited or spontaneous
mutations in the very large dystrophin gene (2.4 Mb)
(Koenig et al. 1988). Specific mutations result in lim-
ited expression of truncated dystrophin, as in the less
severe Becker muscular dystrophy (BMD), or, in
DMD, complete abolishment of its expression (Koenig
et al. 1989). Dystrophin serves a structural role in
muscle by linking the cytoskeletal protein actin to the
extracellular matrix. This linkage provides critical
support to the muscle’s contractile force and, thus,
patients with DMD are highly susceptible to contrac-
tile-based injury and experience continuous cycles of
myofiber death and regeneration in affected muscles
(Ervasti and Campbell 1993; Petrof et al. 1993). The
mdx mouse model has been used extensively for the
study of gene therapy for DMD (Lynch et al. 2000).
While this model appears to reflect the diaphragm
dysfunction of human forms of the disease (Stedman
et al. 1991), the onset and progression of the disease in
583
skeletal muscle is significantly slower than is observed
in human DMD (De la Porte et al. 1999). Transgenic
mdx mouse studies have established that muscle spe-
cific expression of full length or truncated forms of
dystrophin can prevent the development of DMD
(Lynch et al. 2000) and prompted the investigation of
gene replacement therapy for treating the disease.
Two major obstacles to DMD gene therapy are the
need for widespread transduction of most muscle and
the packaging size limits of most vector systems. As has
been described in Muscle-directed gene transfer for
hemophilia B, AAV serotypes 1, 6, 8, and 9 appear most
efficient at transducing muscle. Studies of AAV gene
transfer for DMD using high-pressure infusion instead
of intramuscular injection have demonstrated significant
improvements in muscle transduction levels. Indeed in
isolated limb studies, high-pressure arterial (Gonin et al.
2005) or venous (Su et al. 2005) infusion of AAV sero-
types transduced most of the muscle. Alternatively,
certain AAV serotypes have emerged superior at trans-
ducing muscle following systemic delivery. AAV-6 vec-
tors were shown to transduce most muscles including all
of the heart and diaphragm with VEGF co-infusion
causing significant increases in transduction efficiency at
lower vector doses (Gregorevic et al. 2004). Recently,
self-complementary AAV-8 vectors were reported to
achieve total systemic striated muscle transduction fol-
lowing intravenous administration of the vector (Wang
et al. 2005c; Zhu et al. 2005). The systemic delivery of
AAV vectors for DMD will certainly require the use of
muscle-specific expression cassettes to control unwanted
gene expression in other tissues (Cordier et al. 2001).
While AAV tropism for muscle makes it a logical
choice to treat DMD, the small packaging capacity of
AAV prevents its use for full length gene replacement
therapy. Yet, the observation that individuals with
large truncations in dystrophin can experience a milder
form of the disease (BMD) combined with dystrophin
mutational analysis have led to the identification of
truncated forms that retain function in vivo (Blake
et al. 2002; Ehrenpreis et al. 1991; Harper et al. 2002;
Koenig et al. 1989; Koenig and Kunkel 1990). Micro-
dystrophins that contain a minimum of four spectrin-
like repeats of the central rod domain are readily
packaged in AAV vectors and significantly improve
muscle function in mdx mice (particularly following
delivery to young animals) following systemic or
intramuscular AAV vector delivery (Abmayr et al.
2005; Dickson et al. 2002; Fabb et al. 2002; Gregorevic
et al. 2004; Harper et al. 2002; Liu et al. 2005c;
Sakamoto et al. 2002; Wang et al. 2000a, 2005c;
Watchko et al. 2002; Yoshimura et al. 2004; Yuasa
et al. 2002; Yue et al. 2003; Zhu et al. 2005). Gener-
ally, diaphragm dysfunction was improved to a greater
extent than limb muscles with micro-dystrophins.
It appears that larger dystrophin fragments may be
more effective at treating dystrophic skeletal muscle,
as slightly larger, mini-dystrophin fragments (>6 kb)
have been reported to be more efficacious at treating
584
skeletal muscle dysfunction in DMD models. Since
mini-dystrophins are outside the packaging limit of a
single AAV vector, an alternative AAV-based ap-
proach for expressing mini-dystrophins has been
developed that employs splicing to generate the larger
mini-dystrophin transcript (Duan et al. 2001; Lai et al.
2005a). This dual AAV vector approach employs one
expression cassette for a splice donor fragment of
dystrophin with the second expressing the splice
acceptor of the same portion of dystrophin (Fig. 3b).
Co-administration of these vectors to mdx mice re-
sulted in successful expression of the mini-dystrophin
with improvements in skeletal muscle function (Lai
et al. 2005a). The accumulation of spliced message
appears to be the major limiting factor to this strategy
(Xu et al. 2004).
While most DMD cases arise from large mutations
that generate frame-shifted mRNA, some are caused by
point mutations or small insertions/deletions that affect
a limited number of exons (Blake et al. 2002; Buzin et al.
2005; Feng et al. 2002a, b; Mendell et al. 2001). As a
result, a novel strategy has been proposed for treating
DMD with AAV gene therapy designed to perturb nu-
clear RNA processing in order to rescue the transcript
by skipping mutant exons (Aartsma-Rus et al. 2004;
Gebski et al. 2003; Goyenvalle et al. 2004; Lu et al.
2003a, 2005; Matsuo 2002; Wilton et al. 1997). High-
pressure femoral artery injection of AAV-1 expressing a
modified U7 small nuclear RNA gene targeting exon 23
in mdx mice restored levels of a truncated dystrophin
protein and prevented contraction-induced injury.
In addition to AAV gene transfer for treating DMD,
a congenital, autosomal recessive MD associated with
defective laminin-alpha2 function has been successfully
treated following muscle-directed AAV gene transfer of
novel miniagrin genes to neonatal mice (Qiao et al.
2005).
Taken together, these preclinical studies illustrate
that treating DMD with AAV gene therapy is feasible
(Table 1). The clinical evaluation of these treatments
will ultimately determine the optimal serotype, delivery
method, and engineered transgene cassettes required to
impact human forms of the disease.
AAV gene therapy in the nervous system
Analysis of AAV serotypes in the nervous system has
revealed that all are highly neurotrophic and capable
of high levels of long-term gene expression following a
single injection in the brain, spinal cord, and dorsal
route ganglia. This has suggested the employment of
AAV gene transfer for treating a wide range of neuro-
logical diseases. Since these disorders affect regions of
the brain, the choice of an AAV serotype in particular
applications depends on the levels of serotype-specific
co-receptors and the volume of vector distribution in the
targeted area. AAV-2 has been reported to efficiently
transduce neurons in the substantia nigra (Burger et al.
2004; Paterna et al. 2004), septum (Landgraf et al. 2003;
Mandel et al. 1999b), globus pallidus (Burger et al. 2004;
Tenenbaum et al. 2000), striatum (Burger et al. 2004;
Dass et al. 2005), and the hilar region of the hippo-
campus (Burger et al. 2004). AAV-1 and AAV-5 vectors
achieve significantly higher distribution and numbers of
transduced neurons than AAV-2 in these areas including
transduction of all regions of the hippocampus (Ahmed
et al. 2004; Burger et al. 2004; Davidson et al. 2000;
Passini et al. 2003; Wang et al. 2003). Interestingly,
AAV-4 appears to exclusively transduce ependymal cells
in the brain (Davidson et al. 2000; Liu et al. 2005a) and
may therefore offer a selective advantage in ependymal
targeted therapies. While AAV-8 transduction efficiency
has been examined in only a few structures, like in many
other tissues, this serotype achieved the highest level of
gene expression (Klein et al. 2006).
The clinical utility of AAV vectors in the central
nervous system would be significantly enhanced by the
ability to achieve more widespread distribution of the
vector (see Table 1 for a summary of targeted diseases).
AAV-2 gene transfer is most diffusion limited due to
characteristics of the vector particle and the small
injection volumes used. Since some neurological disor-
ders will require more widespread gene expression to
achieve clinical improvement, alternative AAV delivery
methods are being investigated. Multiple brain injections
is one approach that would allow a larger number of
neurons to be transduced with AAV vectors. However,
the existence of immune responses to the vector particle
or transgene product is of significant concern with this
strategy (Mastakov et al. 2002b). A study on the re-
administration of AAV vectors has revealed that the
brain is capable of a strong serotype-specific immune
response to the vector particle resulting in lower gene
expression and cytotoxicity (Peden et al. 2004). Prior
immunization of rodents with AAV-2 particles resulted
in severe defects in transduction efficiency with neuronal
cell death following another round of AAV-2 vector
delivery. This effect was not observed when AAV-5
vectors were administered following AAV-2 suggesting
that multiple injections of different serotypes may be
feasible. Physical methods to increase the distribution of
AAV-2 vectors in the brain have also been reported. Co-
infusion of AAV-2 with soluble heparin sulfate and/or
fibroblast growth factor (Hadaczek et al. 2004; Masta-
kov et al. 2002a) has been reported to increase transgene
distribution presumably by binding available cellular
AAV-2 co-receptors near the injection site. Convection-
enhanced delivery methods involving the extremely slow
infusion of AAV vectors significantly increased the
effective transduction volume in the brain (Bankiewicz
et al. 2000; Hamilton et al. 2001). Finally, induction of
central hyperosmolarity with central or systemic
administration of mannitol prior to AAV vector brain
injections resulted in better distribution of gene transfer
(Burger et al. 2005; Fu et al. 2003; Mastakov et al. 2001).

Gene therapy for neurological disease
The AAV gene therapy for treating neurological disease
is currently being assessed in a wide variety of toxin-
induced and transgenic rodent models. Generally, AAV-
mediated delivery of neurotrophic factors to slow the
progressive death of neuronal subtypes or transgenes
designed to eliminate/substitute for a dysfunctional
mutant proteins associated with the disease are being
investigated.
Alzheimer’s disease
Alzheimer’s disease (AD) is a neurodegenerative disor-
der characterized by progressive loss of memory, judg-
ment, and language skills. The exact cause of the disease
is not known, but is associated with the accumulation of
amyloid plaques and neurofibrillary tangles throughout
the brain. This results in synaptic loss and cell death in
most cortical structures. Since such a large portion of the
brain is affected, AAV gene therapy for AD has relied
on the use of secreted transgenes or vaccine-based
approaches to be therapeutic. AAV transfer of nerve
growth factor (NGF) has been investigated based on its
documented improvements in cholinergic function and
memory. Mandel et al. (1999b) reported that AAV
delivery of NGF to the medial septum protected cho-
linergic neurons in a transection model of cell death.
Similar improvements were reported by Klein et al.
(2000) after NGF expression following AAV delivery to
the basal forebrain protected against ageing-induced
decreases in size of cholinergic neurons and memory
loss. This approach is limited by the fact that only one
neuronal subtype relevant to AD pathology is targeted.
An alternative vaccine-based strategy with potential for
brain-wide effectiveness has involved intramuscular or
oral delivery of AAV vectors that express amyloid in
order to generate amyloid-specific antibodies (Hara
et al. 2004). This approach eliminates the requirement of
widespread neuronal transduction for total correction
of AD and has resulted in global reductions in brain
plaques without inflammation.
Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is a fatal neurode-
generative disorder that affects the alpha-spinal and
facial motor neurons. After initial muscle weakness,
patients progress to paralysis and respiratory failure
within 5 years. In addition to toxin-induced models of
the disease, gain of function mutations in Cu/Zn
superoxide dismutase (SOD1) has been identified in
familial forms of ALS that have led to the development
of transgenic models.
The AAV delivery of siRNA targeting an activating
mutation in SOD1 (G93A) resulted in a significant de-
585
crease in SOD1 mRNA levels and increased the grip
strength in the injected limb prior to disease onset
(Miller et al. 2005). The AAV delivery of neurotrophic
factors has also resulted in improved motor function in
this mouse model. Intramuscular injection of AAV/
GDNF into all limbs resulted in retrograde transport of
GDNF to motor neuron cell bodies and delayed the
onset of the disease (Lu et al. 2003b; Wang et al. 2002b).
Similar results were achieved following AAV-2 delivery
of GDNF or IGF-1 to hind limbs or intercostals muscles
of G93A mice. In this study, improvements in survival
were determined to be dependent on retrograde trans-
port of intact virus (Kaspar et al. 2003). IGF-1 delivered
at disease onset can also be improved in association with
exercise (Kaspar et al. 2005).
Intraspinal AAV-mediated delivery of anti-apoptotic
gene has also been investigated for the treatment of
ALS. The delivery of the Bcl-2 gene directly to the spinal
cord improved motor neuron survival and motor func-
tion in a transgenic ALS model (Azzouz et al. 2000).
Canavan’s disease
Canavan’s disease (CD) is a monogenetic disorder
characterized by the accumulation of N-acetyl-aspartate
(NAA) in the brain due to the absence of aspartoacylase
activity (ASPA). This results in macrocephaly and
generalized myelin vacuolation leading to severe motor
and cognitive impairment with most patients dying by
2 years of age. A clinical trial using AAV-ASPA gene
replacement therapy was approved for CD patients prior
to preclinical testing (Table 1) (Janson et al. 2002). Since
then, transgenic rodent models have been developed that
lack ASPA with conflicting results following AAV-
ASPA gene replacement observed. While AAV delivery
of ASPA to the striatum and thalamus of CD mice
resulted in high levels of ASPA expression, reduction in
the level of NAA, and improvement in tests of balance
and coordination (Matalon et al. 2003), another study
using a transgenic rat model reported no benefit from
AAV-ASPA therapy (Klugmann et al. 2005a; McPhee
et al. 2005).
Epilepsy
Epilepsy is characterized by seizures that are classified
depending on the site of origin and their recurrence.
Familial forms of the disease have been described and
involve mutations in ion channels that determine neu-
ronal excitability. Treatment strategies currently being
investigated in preclinical epilepsy models have at-
tempted to block excitation by inhibiting the function of
excitatory neurotransmitters or otherwise by modifying
neuronal excitability (Vezzani 2004).
Transgenic mice that lack expression of galanin or its
receptor experience spontaneous seizures. AAV has been
utilized to deliver galanin to the hilar region of the
586
hippocampus in a toxin-induced mouse seizure model.
Galanin overexpression was reported to prevent cell
death in hilar neurons in one study and reduce the
severity of seizures in another (Haberman et al. 2003;
Lin et al. 2003). Another neuroactive peptide, neuro-
peptide Y (NPY), has also been delivered in epilepsy
models using AAV vectors (Richichi et al. 2004). NPY
levels increase following seizure and overexpression of
NPY or one its receptors can inhibit seizures. This effect
is apparently the result of an inhibition of glutamate
release. The AAV-1/2 chimeric vector-mediated delivery
of NPY in the hippocampus reduced seizure severity
by delaying their activity and increasing the threshold
required for convulsions.
Oral immunization of rats with AAV expressing the
NR1 subunit of the excitatory NMDA receptor resulted
in the generation of autoantibodies and ultimately
reduced the neuronal firing of cells that utilize NMDA
receptors (During et al. 2000). A significant reduction
in epilepsy-induced hippocampus damage in a toxin-
induced epileptic model was reported. Finally, a study
involving the AAV gene transfer of homer1a protein to
the hippocampus prevented entry into status epilepticus
(Klugmann et al. 2005b).
Huntington’s disease
Patients with the autosomal dominant Huntington’s
disease (HD) have severe cognitive, psychiatric, and
motor dysfunction. HD is caused by the expansion of
glutamine (Q) codons in the first exon of the HD gene,
resulting in poly-Q expansions in the gene product,
huntingtin. Expansions of greater than 38 glutamine
repeats in huntingtin cause HD, while lower than 35 do
not. The age of onset of this fatal disease is reduced with
higher numbers of repeats. While the endogenous role of
huntingtin and the exact pathological mechanisms of
mutant huntingtin are not known, mutant forms
aggregate with selective loss of GABA-ergic spiny neu-
rons in the striatum (Ravikumar et al. 2002; Scherzinger
et al. 1999). Transgenic mouse models and excitotoxin-
induced models of HD are currently being employed to
study AAV gene therapy for HD.
Preventing striatal neuron death using AAV vectors
for the delivery of neurotrophic factors or elimination of
mutant huntingtin is currently being pursued. Intrastri-
atal injection of AAV vectors expressing brain-derived
neurotrophic factor (BDNF) or glial-derived neuro-
trophic factor (GDNF) (Kells et al. 2004; McBride et al.
2003) has been reported to protect against striatal neu-
ronal loss in two toxin-induced models of HD. While
promising, the possibility for unwanted side effects of
these factors may limit this approach until tightly reg-
ulated expression cassettes can be effectively employed.
Furthermore, efficacy of BDNF and GDNF in trans-
genic HD models with poly-Q expansions in huntingtin
remains to be determined. The endogenous role of
huntingtin has not been elucidated. However, it is clear
that the mutant form of huntingtin has detrimental
effects on neurons. Treatment strategies that eliminate
the mutant huntingtin while preserving the function of
the normal allele are desired. AAV-mediated delivery of
shRNA targeting mutant huntingtin has been developed
(Harper et al. 2005). Injection of AAV-1 or AAV-5
shRNA vectors into the striatum of transgenic HD mice
alleviated both the cellular and behavioral phenotype of
the disease by reducing mutant huntingtin by 50%
(Rodriguez-Lebron et al. 2005).
Targeting CNS for treatment of lysosomal storage
disorders
Gene transfer to the brain is also of great significance for
the treatment of lysosomal storage disorders with CNS
manifestations. As an example, treatment of MPS VII is
discussed in the following. In order to reduce storage in
the brain, vector expressing GUSB may be injected
directly into the parenchyma or administered into the
cerebrospinal fluid by intrathecal injection. The latter
method resulted in sustained (several months) partial
correction of lysosomal storage in the brains of MPS VII
mice after a single injection of AAV-2 vector in adults or
neonates (Elliger et al. 1999; Passini and Wolfe 2001;
Watson et al. 1998). Following direct injection into the
parenchyma of the brain, it was noted that local gene
transfer could result in cross-correction of distal cells
(Sferra et al. 2000; Skorupa et al. 1999). Likely facili-
tated by this cross-correction, not only reduction in
storage, but also improvements in cognitive function (as
determined by the Morris water maze test) were
achieved (Frisella et al. 2001). Moreover, neurodegen-
erative lesions typically seen in the hippocampus and
cerebral cortex of adult MPS VII mice were reversed by
AAV-GUSB vector administration (Bosch et al. 2000;
Heuer et al. 2002). Interestingly, unilateral injection of
vector to the hippocampus resulted in enzyme activity in
both hemispheres in those regions that axonally connect
to the site of gene transfer. Thus, axonal transport may
contribute to the distribution of therapeutic protein in
addition to extracellular diffusion of secreted GUSB,
which can be taken up by cells via the mannose-6-
phosphate receptor (Passini et al. 2002). Uptake of
lysosomal storage enzymes may be further increased by
the incorporation of protein transduction domain Tat
from HIV, and transduction of brain parenchyma can
be improved by use of alternative serotypes such as
AAV-1 (Passini et al. 2003; Xia et al. 2001).
Parkinson’s disease
Patients with Parkinson’s disease (PD) experience dev-
astating motor dysfunction due to death of dopaminer-
gic neurons of the substantia nigra that projects into the
striatum (Hornykiewicz and Kish 1987). The progressive
loss of striatal dopamine results in rigidity and tremor.

PD is managed clinically with the dopamine precursor
L-dopa, but after symptomatic relief for several years
the patients relapse and develop side effects to the
drug (Bergmann et al. 1987). Synthesis of dopamine is a
two-step process in which the amino acid tyrosine is
converted to L-dopa by the rate-limiting tyrosine hydr-
oxylase (TH) with ultimate conversion of L-dopa to
dopamine by aromatic amino acid decarboxylase 1. TH
function is critically dependent on the cofactor BH4 for
its function. This factor is produced by the enzyme GTP
cyclohydrolase I.
The employment of AAV vectors for the treatment
of PD has generally focused on preventing the loss of
dopaminergic neurons in the substantia nigra by AAV
gene transfer of neurotrophic factors and AAV-mediated
delivery of dopamine forming enzymes directly to the
striatum (Mochizuki and Mizuno 2003).
In addition to its ability to prevent the loss of striatal
neurons in models of HD, GDNF is a highly efficacious
growth factor for dopaminergic neurons in the substantia
nigra. In rodent and non-human primate models of PD,
AAV delivery of GDNF to both the substantia nigra and
striatum has proven effective at preventing the loss of
dopaminergic neurons with partial restoration of motor
function (Eslamboli et al. 2005; Mandel et al. 1997, 1999a;
Ozawa et al. 2000; Wang et al. 2002a). AAV delivery of a
related neurotrophic factor, neurturin, is also being
investigated in PD models. Importantly, neurturin
appears as efficacious as GDNF in these models and lacks
the side effects associated with the robust overexpression
of GDNF (Horger et al. 1998; Rosenblad et al. 1999).
Another strategy being investigated for AAV gene
therapy of PD involves the production of dopamine
directly at its site of action. Normal levels of TH and
BH4 are significantly lower in the striatum compared to
the substantia nigral neurons. Therapeutic benefits in
PD models following AAV-mediated transfer of TH
and/or GTPCH1 are currently being investigated with
co-delivery of TH and GTPCH1 resulting in the highest
levels of striatal dopamine and alleviation of PD
symptoms (Kirik et al. 2002; Leff et al. 1999; Mandel
et al. 1998; Muramatsu et al. 2002; Sanchez-Pernaute
et al. 2001; Shen et al. 2000).
Gene therapy for cancer
Cancer can arise in cells of any tissue after mutation of
genes related to cellular proliferation and apoptosis are
acquired. The result can be uncontrolled cell division
and the ability of the malignant cell to metastasize
and invade other organs. A better understanding of the
molecular aspects of cell cycle regulation, apoptosis, and
tumor–host relationships has resulted in the investiga-
tion of diverse anticancer gene therapies using AAV
vectors. In general AAV has been employed in anti-
cancer strategies designed to target the cancer cell
directly or manipulate host mechanisms of tumor
immunity and angiogenesis.
587
The low immunogenic and mutagenic potential of
AAV vectors suggests their application in cancer gene
therapy would be safe and may be beneficial. Further-
more, AAV vectors are able to transduce both dividing
and non-dividing cell types that are typically found in
different areas of the solid tumor. While significant
anticancer effects have been reported using AAV vec-
tors, one potential limitation to their use in cancer gene
therapy is the slow process of second-strand synthesis to
generate a transcriptionally active template. The appli-
cation of self-complementary AAV vectors demon-
strated to achieve high transgene levels immediately
upon viral uncoating should prove valuable in treat-
ments where rapid gene expression is required (McCarty
et al. 2001, 2003; Xu et al. 2005). In addition, the study
of newer AAV serotypes with higher transduction effi-
ciency than AAV-2 is clearly warranted (Gao et al.
2002).
Cancer gene therapy targeting the tumor cell
Tumor suppressors
Loss of function mutations in genes normally involved
in restricting cell cycle progression is one gene replace-
ment strategy using AAV-2 vectors that has shown
promise in preclinical cancer models. For example, the
AAV2-mediated transfer of wild-type p53 to tumors
harboring mutant p53 resulted in cell cycle arrest and
apoptotic death (Qazilbash et al. 1997). Similarly, tumor
growth delay and long-term survival were achieved fol-
lowing AAV-2 transfer of the fragile histidine triad tu-
mor suppressor to human pancreatic tumor xenografts
(Dumon et al. 2001a, b). In a related approach, HPV-
positive cervical tumor growth delay was achieved
following intratumoral delivery of AAV-2 vectors
expressing monocyte chemoattractant protein-1 known
to suppress the function of HPV E6/E7 oncogenes
(Kunke et al. 2000). Finally, AAV2-mediated expression
of maspin, a serine protease often down-regulated in
prostate cancer, resulted in significant tumor growth
delay in vivo (Watanabe et al. 2005).
Enzyme/prodrug
Tumor-selective bio-activation of a pro-drug to a toxic
species following intratumoral AAV-2 delivery of the
activating enzyme is also being investigated. Utilizing
the well-characterized delivery of herpes simplex thy-
midine kinase gene (HSV-tk) followed by treatment with
gancyclovir, AAV2-HSVtk gene delivery has proven
effective in treating a variety of human tumor xenograft
models including hepatoma, glioma, oral squamous
carcinoma, and sarcoma (Berlinghoff et al. 2004; Fukui
et al. 2001; Mizuno et al. 1998; Okada et al. 1996;
Su et al. 2000; Veldwijk et al. 2004). These studies
involved constitutive HSV-tk expression with ex vivo or
588
intratumoral AAV-2 delivery to achieve selective trans-
duction of tumor cells. Transcriptional targeting is an-
other approach being employed to enhance the tumor-
selective cytotoxicity using promoters from the tissue of
tumor origin. For instance, selective killing of hepato-
cellular carcinoma cells with gancyclovir was reported
with AAV-2 vectors when HSV-tk expression was con-
trolled by an albumin/alphafetoprotein hybrid promoter
(Su et al. 1996, 1997). Similarly, tumor-selective cyto-
toxicity was reported for melanoma tumor cells with
AAV2-HSVtk expression controlled by the melanoma
inhibitory activity promoter (Schoensiegel et al. 2004).
Transcriptional targeting of tumor cells based on higher
levels of glucose metabolism is a related approach that
can achieve proliferating tumor cell selective killing with
AAV2-HSVtk therapy. Tumor cell selective cytotoxicity
has been reported using the glucose transporter isoform
1 promoter to control AAV gene transfer of HSV-tk
(Sieger et al. 2004). Finally, based on AAV-2’s tropism
for tumor cells versus peripheral blood progenitor cell
types, the HSV-tk system has been used ex vivo to
eliminate contaminating tumor cells from hematopoietic
stem cells prior to transplantation (Veldwijk et al. 1999,
2000).
Drug resistance
The development of drug resistance is a major issue
associated with the treatment of cancer with multimo-
dality chemotherapy regimens. AAV-2 vectors are being
investigated to reverse the cellular phenotypes respon-
sible for this loss in drug sensitivity. Self-complementary
AAV-2 vectors have been utilized to overcome drug
resistance associated with the induced expression of the
transmembrane ATPase, p-glycoprotein (Xu et al. 2005).
P-glycoprotein is an effective efflux pump capable of
removing a wide range of chemotherapy agents from
drug-resistant cell types. AAV-2 vectors expressing
p-glycoprotein-targeted siRNA have been used to effec-
tively lower the levels of p-glycoprotein in multidrug-
resistant breast and oral carcinoma cells and sensitize
these cells to chemotherapy (Xu et al. 2005).
Cancer gene therapy targeting the host
Immunotherapy
Studies of natural killer cells and the tumor necrosis
factor and interferon family of cytokines have sup-
ported the existence of tumor immunity. In general,
tumors can illicit strong immune responses during
early tumor stages, but eventually this defense is lost
allowing more aggressive tumor growth. Several
mechanisms have been suggested for this loss of tumor
immunity including weak tumor antigens, low levels of
MHC class II and costimulatory molecules, and the
tumor cell production of general immunosuppressive
factors. AAV-2 vector delivery of dominant epitopes to
tumor or antigen-presenting cells in tumor vaccine
strategies have shown significant efficacy in some
models. AAV-2 delivery of CD40 ligand to B-cells
from chronic lymphocytic leukemia (CLL) patients
resulted in HLA-matched T cells following the induc-
tion of CD80 on CLL cells (Wendtner et al. 2002).
A related approach utilizing systemic expression of
the HPV16-E7 CTL epitope/heat shock fusion protein
effectively immunized animals subsequently challenged
with E7-expressing tumor cells (Liu et al. 2000). While
AAV-2 vectors fail to transduce mature dendritic cells
in vivo, significant antitumor responses have been
reported following the AAV-2 mediated transfer of
HPV-16 oncogenes (Liu et al. 2000), carcioembryonic
antigen (Ponnazhagan et al. 2004b), and BCR-ABL
fusion domains (Sun et al. 2002) to immature dendritic
cells ex vivo. Reinfusion of these transduced cells
elicited a strong CTL cell response and impaired tumor
growth in cervical, colon, and breast cancer models,
respectively.
The delivery of cytokines directly toxic to the tumor
and/or capable of boosting antitumor immunity has also
achieved positive outcomes using AAV-2 vectors. For
example, AAV-2 delivery of tumor necrosis factor-
related apoptosis-inducing ligand inhibited tumor for-
mation in colorectal, lung, and liver tumor models (Ma
et al. 2005a, b; Mohr et al. 2004; Shi et al. 2005; Yoo
et al. 2006). Similar improvements in survival following
AAV-2 delivery of interferon have been reported fol-
lowing ex vivo or intratumoral transduction of cancer
cells (Chen et al. 2005a; Streck et al. 2004a, 2006;
Yoshida et al. 2002; Zhang et al. 1996).
Antiangiogenesis
It is now well established that solid tumors must induce
angiogenesis in order to reach a clinically relevant size,
metastasize, and grow in other tissues. The continual
expansion of the tumor blood vessel network supplies
critical nutritional requirements allowing for progressive
tumor development, growth, and survival. As a result,
targeting angiogenesis and the proliferation of endo-
thelial cells has emerged as a promising treatment
strategy for all tumor types.
The AAV2-mediated systemic expression of antian-
giogenic transgenes has shown high potency in a
number of tumor models using different sites for vec-
tor injection. High levels of angiostatin expression and
improvements in tumor-free survival were achieved in
a human glioma model following injection of AAV-2
intramuscularly or intratumorally (Ma et al. 2002a, b).
Similarly, AAV-2 angiostatin vectors administered via
portal or tail vein injection also suppressed tumor
growth and prolonged survival in various preclinical
tumor models (Isayeva et al. 2005; Lalani et al. 2004;
Xu et al. 2003).

Similar antitumor responses have been reported
following AAV-2 delivery of the related antiangiogenic
peptide, endostatin. Shi et al. (2002) demonstrated that
therapeutically relevant levels of endostatin could be
achieved following intramuscular administration of
AAV vectors that was accompanied by impairment of
tumor vessel formation and tumor initiation of xeno-
grafted human colorectal tumor cells. This group went
on to show that systemic expression of endostatin
enhanced the response of these tumors to ionizing
radiation (Shi et al. 2003). In a related study, intra-
tumoral AAV-2 delivery of endostatin to hepatocellu-
lar carcinoma cells significantly suppressed the tumor
growth in vivo (Liu et al. 2005b); and tumor growth
suppression and impairment of metastasis were re-
ported in an orthotopic pancreatic cancer model in
hamsters following intraportal vein AAV-2 endostatin
gene therapy (Noro et al. 2004). Recently, the delivery
of a mutant form of endostatin, engineered to bind
more effectively to endothelial cells, inhibited the
growth of human ovarian cancer xenografts (Subra-
manian et al. 2005).
Other antiangiogenic transgenes have demonstrated
efficacy after AAV-2 gene delivery. AAV2-mediated
systemic expression of a dominant negative VEGF-2
receptor in the liver suppressed the development of renal
tumor xenografts (Davidoff et al. 2002, 2005b; Streck
et al. 2004b). In the first gene therapy approach for
delivery of monoclonal antibodies, high levels of sys-
temic expression of a neutralizing antibody for the
VEGF-2 receptor resulted in significant antitumor effi-
cacy following a single administration of AAV-8 (Fang
et al. 2005).
Finally, AAV-2 vectors have also been employed to
inhibit the metastatic and angiogenic potential of cancer
cells by inhibiting their ability to invade tissues. Signif-
icant suppression of Kaposi’s sarcoma growth was
reported following intratumoral delivery of a tissue
inhibitor of metalloproteases using AAV-2 vectors
(Zacchigna et al. 2004).
Multimodality cancer gene therapy
Most successful anticancer treatments involve the use of
multiple agents with non-overlapping normal tissue
toxicities. The lack of toxicity of AAV vectors per se
makes them well suited for inclusion in these multimo-
dality regimens. Furthermore, the reported enhancement
in AAV transduction efficiency following the treatment
of cells with DNA damaging agents illustrates that such
combinations may be beneficial. For example, ionizing
radiation and topoisomerase inhibitors are commonly
used in the clinic to treat cancer and significantly
increase transgene levels in cells infected with AAV
vectors (Alexander et al. 1994; Russell et al. 1995).
Taking advantage of this interaction, a report combin-
ing ionizing radiation and topoisomerase inhibitors
with AAV2-HSVtk cancer gene therapy reported higher
589
levels of HSV-tk gene expression and cytotoxicity
following treatment of hepatoma and ovarian tumors
with gancyclovir (Kanazawa et al. 2003, 2004). Similar
enhancement in antitumor response was reported with
AAV-2 endostatin delivered to the tumor cells in com-
bination with topoisomerase inhibitors (Hong et al.
2004). Newer classes of compounds with anticancer ef-
fects currently under development such as proteasome
and HDAC inhibitors have also been reported to en-
hance AAV transduction efficiency in some cancer cell
types (Hacker et al. 2005; Okada et al. 2005). In vivo
analysis of tumor responses to these combinations may
reveal similar improvements to AAV gene transfer and
help elucidate mechanisms of AAV transduction in the
cancer cell.
The expression of multiple transgenes is another
AAV gene therapy approach being investigated for
treating cancer. Since endostatin and angiostatin impair
tumor-induced angiogenesis through different receptor
mechanisms, the AAV-2 delivery of these peptides in
combination achieved synergistic antitumor responses
in vivo (Ponnazhagan et al. 2004a). Combining an-
giostatin gene transfer with the B7.1 apoptosis-inducing
ligand was also more beneficial than either alone (Sun
et al. 2005c). Significant improvements on AAV-HSVtk
responses have been reported when combined with AAV
delivery of B7.1 (Janouskova et al. 2003), IL-2 (Su et al.
2000), GMCSF (Janouskova et al. 2003), and MCP-1
(Janouskova et al. 2003) transgenes.
AAV vector targeting for cancer gene therapy
Cancer gene therapy often involves the delivery of toxic
transgenes to malignant cells. Critical to the safe and
effective use of these approaches is the development of
vector systems that selectively target these transgenes to
the tumors. In addition to the intratumoral delivery
methods and the use of tissue or tumor-specific pro-
moter elements described above, modification of AAV
vector tropism through manipulation of the capsid
proteins is being employed to target cell surface recep-
tors selectively overexpressed on tumor cells or prolif-
erating tumor vasculature. Incorporation of the amino
acid sequence, NGRAHA, into the heparin-binding
motif of the AAV-2 capsid retargeted the vector to cell
surface CD13 expression on Kaposi’s sarcoma and
rhabdomyosarcoma tumors cells as well as proliferating
endothelial cells (Grifman et al. 2001). Similarly retar-
geting AAV to integrin receptors has been developed to
target these cell types. Insertion of the integrin-binding
RGD sequence at the same position in AAV-2 vectors
provided a heparin-independent method of vector entry
into endothelial and ovarian tumor cell lines (Shi et al.
2001; Shi and Bartlett 2003). Finally, the insertion of the
amino acid sequence, SIGYPLP, into the AAV-2 hep-
arin-binding motif allowed AAV transduction of previ-
ously refractory tumor cell lines (Nicklin et al. 2001,
2003).
590
Gene therapy for obesity
Obesity rates have increased significantly worldwide in
recent years. Significant increases in adipose tissue have
been shown to increase the risk of developing many
conditions including coronary heart disease, hyperten-
sion, and type 2 diabetes. The lack of an effective
pharmacological solution for treating obesity has re-
sulted in the investigation of central and peripheral AAV
gene therapy for reducing adiposity in transgenic and
diet-induced obesity models. In general, gene therapy
treatments for obesity have focused on the delivery of
hormones and neurocytokines that can decrease food
intake and/or increase energy expenditure (Zolotukhin
2005).
Leptin is a hormone produced predominately by
adipose tissue and acts centrally in the hypothalamus to
reduce food intake and increase energy expenditure.
Intramuscular administration of AAV vectors for sys-
temic expression of leptin demonstrated long-term cor-
rection of obesity and associated type II diabetes in
genetically obese mice (Murphy et al. 1997). However,
detrimental side effects of leptin overexpression in the
peripheral circulation were observed. Subsequently,
hypothalamic injection of AAV expressing leptin re-
sulted in significantly higher therapeutic responses
without peripheral side effects (Beretta et al. 2002;
Dhillon et al. 2001; Dube et al. 2002; Lundberg et al.
2001; Scarpace et al. 2002b). AAV leptin gene therapy is
initially quite potent, but resistance to leptin gene
delivery often develops (Scarpace et al. 2001, 2002a,
2005; Shek and Scarpace 2000). Therefore, alternative
peptides that activate similar transduction cascades to
those induced by leptin receptor occupancy are being
investigated for use in AAV gene therapy. Ciliary neu-
rotrophic factor (CNTF) and leukemia inhibitory factor
(LIF) are IL-6 family neurocytokines that have been
employed in AAV gene transfer to the hypothalamus.
Constitutive expression of these peptides centrally re-
sulted in temporal weight loss (CNTF) or severe ca-
chexia (LIF) (Prima et al. 2004). While these peptides
could treat obesity in leptin-resistant models, the short-
lived action and severe side effects should limit these
cytokines clinically.
The central melanocortin system is comprised of
various melanocortin peptides that are critical regulators
of feeding and body weight (Zhang and Scarpace 2006).
Melanocortins, such as alpha-melanocortin, are derived
from a common precursor, pro-opiomelanocortin, and
reduced hypothalamic expression of this protein is
associated with obesity syndromes such as leptin resis-
tance and aging. Hypothalamic gene delivery of
pro-opiomelanocortin with AAV vectors resulted in
hypophagia, reduced adiposity, and improved insulin
sensitivity in a genetically obese, leptin-resistant rat
model (Li et al. 2003). Similarly, glucose intolerance and
onset of age-related obesity-associated impaired hypo-
thalamic expression of pro-opiomelanocortin could be
improved following hypothalamic AAV gene transfer of
pro-opiomelanocortin (Li et al. 2005).
Adiponectin is a hormone secreted by adipose tissue
that is generally lower in obese models and upon
replenishment has the ability to reduce adiposity and
overcome the associated insulin resistance in rodent
obesity models. Adiponectin acts peripherally by
increasing fatty acid oxidation in muscle and liver, while
decreasing hepatic glucose production. Systemic
expression of adiponectin after AAV gene transfer to
muscle and liver can prevent development of diet-in-
duced obesity and ameliorated insulin resistance for at
least 41 weeks in a diet-induced obesity model (Shklyaev
et al. 2003).
Gene therapy for autoimmune disease
Autoimmune diseases are characterized by the develop-
ment of inflammatory conditions due to activation of
auto-reactive T cells and antibodies and the expression
of pro-inflammatory cytokines. The central goal of AAV
gene therapy for these conditions is to shift the balance
to a non-inflammatory phenotype by expressing
transgenes to impart tolerance to the self-antigen or
antagonize the inflammatory cytokines (Evans et al.
2004; Gould and Favorov 2003; Tarner and Fathman
2002). These strategies have utilized systemic expression
of therapy by intramuscular administration of AAV
vectors or the delivery of vector directly to the affected
tissue.
Diabetes
Type I diabetes results from an autoimmune destruction
of pancreatic beta cells leading to the loss of insulin
production. AAV gene therapy for diabetes has shown
disease amelioration in streptozocin-induced and a non-
obese diabetic (NOD) congenic mouse models. In
particular, systemic overexpression of the immunosup-
pressive cytokine interleukin-10 (IL-10) helps prevent
destruction of islet cells in NOD mice if given prior to
disease onset (Goudy et al. 2001). Islet transplantation is
an alternative therapy being investigated to restore glu-
cose homeostasis in type 1 diabetes (Carter et al. 2004,
2005; Flotte et al. 2001). However, persistent autoim-
munity and allogenic immunities limit clinical success.
NOD mice newly diagnosed with diabetes also benefit
from muscle-directed AAV IL-10 therapy given in
combination with islet cell transplants with increases in
regulatory T cells, reduced lymphocyte infiltration, and
induced antioxidant enzymes in islet grafts (Goudy et al.
2003).
Similar reduction in diabetes pathology has been re-
ported following AAV-mediated delivery of the anti-
inflammatory, a1-antitrypsin gene in NOD mice (Song
et al. 2004). AAV vectors have also been utilized to in-
duce tolerance of auto-reactive T cells to pancreatic beta

cell auto-antigens. AAV delivery of the dominant anti-
genic epitope of glutamic acid decarboxylase 65 signifi-
cantly reduced the severity of the disease in vivo (Han
et al. 2005). Finally, an alternative strategy being
developed for treating diabetes involving the regulated
expression of system insulin levels utilizing AAV hepatic
gene transfer of the preproinsulin B10 gene under con-
trol of the glut2 promoter has resulted in the reduction
of hyperglycemia in streptozocin-induced diabetes
(Burkhardt et al. 2005).
Arthritis
Adeno-associated viral vectors have been shown to
readily transduce synoviocytes and primary chondro-
cytes in vitro (Arai et al. 2000; Jennings et al. 2005).
Studies of AAV gene transfer directly to joints have
demonstrated persistent gene expression with AAV-5
exhibiting higher transduction efficiency than AAV-2
vectors (Adriaansen et al. 2005; Apparailly et al. 2005;
Madry et al. 2003; Watanabe et al. 2000). Transgenic
rodent models of arthritis have facilitated the elucida-
tion of key mediators of inflammation and cartilage loss
in arthritis and have been used to study the application
of AAV gene therapy to diseased joints. Disease-induced
gene expression using AAV vector has been observed
dependent on the promoter employed for controlling
gene expression. CMV promoter-driven inflammation-
enhanced transduction of synovial cells has been ob-
served after reactivation of silenced gene expression (Pan
et al. 1999). Tumor necrosis factor alpha and interleu-
kin-1 (IL-1) appear to play significant roles in arthritis.
As a result AAV gene therapy for arthritis has initially
focused on antagonism of the effects of these cytokines
in the arthritic joint. AAV-mediated expression of an
IL-1 receptor antagonist (IL1Ra) in the joint significantly
improved disease markers and could be reactivated
preventing recurrent arthritic episodes (Pan et al. 2000).
Similarly, intraarticular AAV gene transfer of a dominant
negative form of the TNF alpha receptor alleviated dis-
ease symptoms following transduction of synoviocytes
and muscle (Chan et al. 2002; Zhang et al. 2000). Intra-
muscular delivery of IL-4 led to systemic overexpression
and injection into tarsus improved indicators (Cottard
et al. 2000). Finally, antiangiogenic therapy using AAV
delivery of angiostatin could prevent collagen-induced
arthritis in mice (Takahashi et al. 2005).
Sjogren’s syndrome
Sjogren’s syndrome (SS) is an autoimmune disorder that
affects the lacrimal and salivary glands resulting in se-
vere eye and mouth dryness. Little is known about the
pathogenic mechanisms behind SS. NOD mice used in
gene transfer studies for diabetes also have inflammation
of the submandibular and lacrimal glands and provide a
good model for SS gene therapy investigations. AAV-
591
mediated gene transfer to salivary glands has been
shown to improve glandular secretions while establish-
ing salivary glands as efficient mediators of systemic
gene transfer (Voutetakis et al. 2004, 2005; Yamano
et al. 2002). IL-10 delivered to salivary glands and
muscle in NOD mice significantly improved production
of saliva with inflammatory infiltrates reduced only with
local delivery to the salivary gland. Improved blood
glucose and insulin levels were also observed following
salivary gland injection of AAV IL-10 vectors indicating
systemic benefit of this delivery method (Kok et al.
2003a, b). Finally, the immunosuppressive effects of
vasoactive intestinal peptide gene transfer to salivary
glands with AAV vectors led to improved salivary flow,
increased salivary gland expression of VIP, and reduc-
tion in salivary gland pro-inflammatory cytokines
(Lodde et al. 2006).
Conclusions
The research described above clearly illustrates the po-
tential of AAV gene therapy to treat a wide range of
human diseases. Still, the clinical application of AAV
gene transfer has revealed many issues that remain for
the safest and most effective therapy using these vectors.
Research efforts in vector technology and immunologi-
cal mechanisms that impact AAV gene transfer are
essential to develop curative therapies. While AAV
serotypes have been identified with significantly higher
transduction efficiency, vector promiscuity remains a
major obstacle to selective expression of transgenes. The
identification of cellular receptors for the different AAV
serotypes and capsid residues that mediate this interac-
tion is essential for the development of vectors with cell
type specific transduction. Furthermore, the use of re-
targeted AAV vectors in combination with tissue-selec-
tive or regulated transgene expression should improve
the overall safety of these treatments. A more thorough
evaluation of the innate and acquired immunity to the
available serotypes and novel hybrid/capsid modified
vectors is also required (Manno et al. 2006; Sabatino
et al. 2005). Indeed, two recent preclinical studies and
the completion of hemophilia B clinical trial have dem-
onstrated that specific CTL responses targeting AAV
capsid protein epitopes limit the duration of therapeu-
tically relevant transgene expression and the potential of
cross-reactivity between epitopes for the different sero-
types (Chen et al. 2006; Sabatino et al. 2005). More
extensive mapping of T cell epitopes in the human
population is required. Strategies (such as immune
modulation/suppression) to eliminate these immune re-
sponses are emerging and may have a profound impact
on the use of AAV gene transfer in the clinic. Finally,
while the integration potential for recombinant AAV
vector genomes is very low, a clearer delineation of
genomic ‘‘hot spots’’ where AAV insertional mutagen-
esis may occur is critical to patient safety (Nakai et al.
2003b, 2005b).
592
Acknowledgements The authors thank Drs. Corrinna Burger, Jef-
frey S. Chamberlain, Cathryn Mah for extensive assistance with the
manuscript.
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