Since the first parvovirus serotype AAV2 was isolated from human
and used as a vector for gene therapy application, there have been
significant progresses in AAV vector development. AAV vectors have been
extensively investigated in gene therapy for a broad application. AAV
vectors have been considered as the first choice of vector due to
efficient infectivity, stable expression and nonpathogenicity. However,
the untoward events in AAV mediated in vivo gene therapy studies
proposed the new challenges for their further applications. Deep
understanding of the viral life cycle, viral structure and replication,
infection mechanism and efficiency of AAV DNA integration, in terms of
contributing viral, host-cell factors and circumstances would promote to
evaluate the advantages and disadvantages and provide more insightful
information for the possible clinical applications. In this review, main
effort will be focused on the recent progresses in gene delivery to the
target cells via receptor-ligand interaction and DNA specific
integration regulation. Furthermore AAV receptor and virus particle
intracellular trafficking are also discussed.

AAV has been considered as a safe vector for gene transfer and has
been involved in many clinical trials to treat metabolic abnormalities,
hemophilia disease, Parkinson's disease and cystic fibrosis. AAV
vectors have following advantages in gene therapy studies. (1) AAV
vector is able to site specifically integrate into the host genome. Site
specific integration of AAV doesn't activate the possible oncogenes
and the inserted gene can be maintained for a relatively long-term in
host cell genome and stably expressed in vivo [55,71,128]. Furthermore,
selective genomic integration can be achieved via genetic modified AAV
vectors [126]. (2) AAV capsids endure capsid modification and serotype
swapping, which allows the AAV-mediated cell targeting delivery based on
virus-host interaction. (3) AAV vectors show wide host spectrum and high
infectivity for dividing and non-dividing cells. (4) AAV vectors also
have advantages for solid cancers' application due to its small
size, because of its high penetrability through the tumor stroma
compared to other viral vectors. (5) AAV vector is low immunogenic. Most
of the AAV serotypes are isolated from human; therefore immune response
against the vector is relatively low and re-administration is possible
due to low in vivo neutralizing antibody. However, AAV vector caused
immune response was discovered in vivo by several groups. Heparin
binding directed activation of cytotoxic T cells against AAV2 capsid,
but not AAV8, was determined, which partially explained the liver
toxicity in AAV2, not AAV8 mediated hemophilia treatment [15,102,53].
Moreover, Wang et al observed AAV2 induced in vivo capsid-specific
cytotoxic T cells, but not AAV7 or AAV8. This CTL exerted the functional
cytolytic effect on capsid-peptide loaded target cells, but not AAV2
vector-transduced hepatocytes [111]. Similarly, AAV2 capsid-specific
cytotoxic T cells only eliminate AAV2 vector transduced cells
co-expressing AAV2 capsid in vivo. Li further pointed out that
additional modification to AAV vectors may be required for further study
for elicitation of cellular immune response [16]. (6) AAV vector is
reported to be non-pathogenic. AAV2 have been reported to be associated
with certain pathogenic processes without causing obvious severe
diseases [64,83]. Some recent studies, however, have shown
hepatocarcinoma induced in AAV-injected mice due to insertion with
6-kilobase region of chromosome 12 [26]. The mechanism and cause for
this phenomenon certainly requires further investigation.

AAV has a broad tissue tropism due to wide expression of AAV
receptors. Targeting may be achieved through local injection to liver,
brain and muscle. However, the most popular and convenient
administration means in human or tested animal models is intravenous
injection. Broad host spectrum limits AAV vector targeting delivery to
specific tissues. Biodistribution study of different serotype AAV
vectors post-intravenous administration showed that AAV DNA was detected
in lymphocyte tissue, particularly in spleen with high frequency and
concentration and also in other tissue including liver, brain, lung,
heart, gallbladder, pancreas, colon, kidney, ovary, uterus etc. [68].
These data indicated the necessity to generate specific gene expression
via AAV vector targeting delivery and to reduce side effect caused by
nonspecific therapeutic gene production. AAV-based gene targeting
therapy strategies can be briefly categorized into the following five
types. (1) Local administration. (2) DNA integration. (3)
Receptor-ligand interaction. (4) Small interference RNA. (5) Tissue
specific promoter. (6) AAV engineered cytotherapy.

AAV receptor and co-receptor: AAV viruses infect the host cells via
the interaction between viral capsid and cell surface. Heparin sulfate
proteoglycan is reported to act as primary receptor for adeno-associated
virus type 2 (AAV-2). Reported AAV2 co-receptors include alphaVbeta5
integrin [94], fibroblast growth factor receptor 1 (FGFR-1) [75] and
hepatocyte growth factor (c-Met) [8]. There was an attachment of AAV-3
strain H to heparin, heparin sulfate and FGFR1 [8,75,94,95]. AAV1 and
AAV3 have different tropism from AAV2, which indicates that these two
serotypes AAV vectors infect cell via different entry. AAV5 has natural
tropism to the lung, central nervous system, muscle and eye. Studies
showed that expression of the platelet-derived growth factor receptor
(PDGFR-alphapolypeptide) was highly correlated with AAV5mediated
transduction. Further investigation confirmed the role of PDGFR-alpha
and PDGFR-beta as receptors for AAV-5. The tropism of AAV-5 in vivo also
correlated with the expression pattern of PDGFR-alpha [24]. Some other
group reported that AAV5 transduction was highly sialic acid dependent;
however, AAV6 was either sialic acid dependent or in-dependent in tested
cells. Moreover, AAV5 didn't inhibit AAV6 mediated gene
transduction, although sialic acid played critical role in their gene
transduction. All these data suggested that AAV5 and AAV6 used different
entry pathway [84]. AAV1 and AAV6 use the alpha2,3 and alpha2,6 sialic
acids that are present on N-linked glycoproteins as primary receptors
[117]. Heparin sulfate can both inhibit AAV2 and AAV3 gene
transductions; however, the ID (50) of rAAV-3 was higher than that of
rAAV-2. Furthermore, virus-binding overlay assays indicated that AAV-2
and AAV-3 bound different membrane proteins, which is the cause that
AAV3 can transduce the haematopoietic cells [40]. The different
transduction efficiency between AAV2 and AAV5 for air way epithelial
cells suggested these two serotype AAV vectors may use the different
receptor. Histological study showed that the apical membrane of airway
epithelia cells generated abundant high affinity receptors for AAV5,
less for AAV2. Genetic or enzymatic removal of sialic acid from the air
way epithelial cell surface significantly inhibited the AAV5 binding and
gene transfer, especially lectin specially binding of 2,3-linked sialic
acid [108]. AAV4 binding required alpha 2--3 O-linked sialic acid,
whereas AAV5 required N-linked sialic acid [107,108]. Soluble alpha 2-3
sialic acid was able to block both AAV4 and AAV5 transduction and alpha
2-6 sialic acid only was able to block AAV5 transduction. A 37/67-kDa
laminin receptor (LamR) was reported to be the cellular receptor for
AAV8 [2]. LamR greatly contributes to AAV8 robust transduction in the
cultured cells and mouse liver in vivo although heparin sulfate also
plays partial role to AAV8 attachment and viral infection. Moreover LamR
plays a role in transduction by three other closely related serotypes
(AAV2, -3 and -9). AAV9 has preferential in vivo transduction in cardiac
tissue compared to AAV8 [46,103]. AAV2, 4 and 5 were confirmed to
transduce the salivary gland cells in vitro and in vivo, however, these
three serotype AAV vectors are different from each other in receptor
binding, indicating they directly attach, bind and enter different cell
types due to the membrane molecule expression variations [49]. There is
no cross-activity among these three AAV serotypes. Taken all together,
there is not only one specific molecule involved as a single receptor
for any serotype AAV virus infection. So far, except AAV2, all the other
serotype AAV receptors still are not very clear. Tropic characteristic
investigation of each serotype can provide important information for
targeting gene transfer.

AAV entry trafficking: AAV virus infection requires the viral
attachment on the cell surface membrane, followed by clathrin-dependent/
independent internalization. Endosome acidification is the critical
process for AAV gene transduction efficiency, which is associated with
early to late endosome transition and proteasome degradation activity
[52]. Bartlett described the detailed AAV2 intracellular trafficking by
covalently conjugating fluorophores to AAV and monitoring entry by
fluorescence microscopy. AAV2 particles were internalized rapidly by
standard receptor-mediated endocytosis from clathrin-coated pits
(half-time <10 min) and viral particle moved to nucleus and
accumulated perinuclearly within 30 min after endocytosis. Furthermore,
AAV particle release after internalization from early endosomes requires
an acidic environment for penetration into the cytosol. Although the
rescue of AAV in the host cell needs helper adenovirus, the escape of
AAV from endosome and trafficking of viral particle to the nucleus are
non-dependent of adenovirus infection [7]. Endosome releasing and
nuclear processing are the rate limitings for gene transfer. However,
Xiao reported that the duration of AAV nucleus translocation from
cytoplasm as intact particles was quickened after co-infection with
adenovirus and this facilitated AAV nuclear entry and translocation was
not blocked by the nuclear pore complex inhibitor thapsigargan,
indicating that one or more adenovirus capsid proteins might be altering
trafficking. Accordant with Bartlett's discoveries, escape from
early endosomes did not seem to be affected by adenovirus co-infection
[118]. Kux genetically incorporated eGFP into the adenoassociated virus
capsid by replacement of wild-type VP2 with GFP-VP2 fusion protein to
allow more specific direct visualization of viral trafficking [59]. The
results showed that nuclear translocation was a slow and inefficient
process compared to adenovirus coinfection, which is consistent with
Xiao's report. GFPtagged AAV particles can be applied for the
studies of viral intracellular trafficking and nuclear entry.
Discoveries obtained through this system argued against an efficient
nulear mechanism of intact AAV capsid and favored the occurrence of
viral uncoating before or during nuclear entry. New studies showed that
Notch1 played a significant role in intracellular trafficking of
recombinant adeno-associated virus type 2 (rAAV2) [80]. In the absence
or low-level expression of Notch1, only binding of virus was found on
the cell surface and internalization was impaired. However, increased
Notch1 expression in these cells allowed efficient perinuclear
accumulation of labeled capsids. Dynamin level were found not to be
different, however, blocking dynamin function abrogated AAV2
transduction in cells over-expressing full-length Notch1 but not those
cells expressing intracellular Notch1.

Targeting gene delivery: Local Administration: Local administration
such as intramuscular, intra-tumor injection and microfusion can be
performed for superficial tumor and tissue specific deficiency. Brain,
muscle and liver are the most easy target tissues for local
administration [22,30,73,112]. Inherited metabolic disease
mucopolysaccharidosis type I (MPS I) was from the deficiency of
alpha-L-iduronidase (IDUA), which is strictly expressed in the brain
tissue. To target gene therapy to the CNS, recombinant AdenoAssociated
Viral (AAV) vectors carrying IDUA sequence were administered to MPS I
mice via injection into cerebrospinal fluid and this intrathecal
administration of AAV-IDUA effectively delivered vector to brain cells
with minimal invasion [112]. Muscle could be another target for local
gene transfer therapy. Hoffman's study showed that gene transfer to
skeletal muscle did not break tolerance achieved by liverderived
expression [44], indicating that muscle has its own privilege as a
target tissue. Subretinal injection obtained the sufficient gene
expression localized in retinal ganglion cells with modified AAV
incorporating a Chicken Beta-Actin (CBA) promoter and the woodchuck
hepatitis posttranscriptional regulatory element[61]. Adeno-associatd
vectors carrying the GFP driven by CBA promoter as a tracer and U6
promoter controlling small hairpin RNA targeting alpha-CaMKII
(AAV-shCAM) were microfused into the rat hippocampus. The localized GFP
expression was achieved and local alpha-CaMKII expression was
significantly reduced as well [74]. Targeting delivery via local
administration can only be achieved in limited situations such as
muscle, skin, air way, brain or joint.

Gene targeting via DNA integration: High frequencies of gene
targeting can be achieved in mammalian cells transduced by recombinant
AdenoAssociated Virus (rAAV) vectors [79,81,119,123,128]. Gene therapy
is to replace the abnormal gene with normal gene to maintain the
sufficient protein production and function in the mutant cells via
non-specific or specific integration. The ideal way is to correct the
mutation via homologous recombination or reverse mutation, allowing the
normal gene expression. Major efforts in the field are aimed towards
targeting vector integration to specific sites in the host genome.
Integration targeting to homologous chromosome sequences has been
observed in vitro and in vivo. AAVS1 locus (19q13-3-qter) was originally
considered as AAV2 integration site [33,51,97]. Studies have shown that
510nt region at the 5' end of AAVS1 DNA was responsible for
directing AAV chromosomal integration signal. Giraud et al. [33] also
indicated that chromosomal integration locus may be involved in the
genomic instability due to the unusual degree of DNA heterogeneity in
the recovered vector associated with AAVS1. Muscle is the most widely
targeted tissue to AAV2 owing to not only local administration but also
genomic DNA targeting. Slow skeleton troponin T gene (TNNT1) is closely
linked with AAVS1 locus and AAV DNA integration in muscle cells results
in TNNT1-AAV junction, suggesting muscle is the natural target tissue
for latent AAV infection [28]. Therefore there are numbers of clinical
trials such as inherited diseases and hemophilia, based on AAV2 mediated
gene transduction in muscle via gene transfer therapy. However,
phenotypic alterations of AAV2-transduced muscle fiber cells may occur
and further investigations are needed. Miller et al introduced AAV2
vector containing LacZ gene fragment into the mouse with
nuclear-localized 4bp-deleted lacZ mutant gene and achieved the precise
correction of LacZ gene [67]. Although there are more progresses in DNA
integration after the studies have been focused on integration via AAV2.
However, this integration might increase the genome instability and
cause the possible oncogene activation and oncogenesis. Miller reported
that integrated AAV vectors were associated with chromosomal deletions
and rearrangements which were frequently located on chromosome 19, while
there were no such instabilities at the wild-type AAV integration site
[66]. In 2004, McCarty pointed out in his review that AAV integration in
any context was inefficient and that the persistence of AAV gene
delivery vectors in tissues was largely attributable to episomal genomes
[63]. Based on widely known conception that Rep78/68 nonstructural
proteins are responsible for AAV DNA integration at Rep Binding Site
(RBS) on AAVS1, recent discovery showed that TRP-185, TRA RNA loop
binding protein, bound to AAVS1 DNA and suppressed AAV DNA integration
at AAVS1 RBS. TRP-185 was shown to change the specificity of DNA
integration from AAVS1 RBS to a downstream region [71]. Human
hematopoietic cells were co-infected with Rep78 and GFP expressing
adenoviruses to study the DNA integration mediated by Rep78. Among all
the analyzed integration sites, 30% vector integrations are at AAVS1 RBS
and 90% sites are mediated by Ad-ITR [113]. Accordantly, Feng et al
found out that there were three Rep Binding Elements (RBE) in the AAV
genome, two are in the inverted terminal repeats and one in newly
discovered region encompassing the viral p5 promoter. RBE located in ITR
was more efficient and specific than p5 RBE in Rep-dependent DNA
integration [13]. AAV integration provides the genomic DNA targeting,
however, the random insertion/integration occurs in parallel. More
specific AAV DNA integration and gene targeting requires the Homologous
Recombination (HR) pathway, as RNAi induced HR-involved gene silencing
abolished stable long-term transgene expression [104]. Homologous
recombination allows efficient, high-fidelity, non-mutagenic gene repair
in a host cell. Integration not only provides the stable, longterm
transgene expression, but also induces human tumor cell phenotype
alteration [109]. AAV DNA integrated HeLa cells have been investigated
for cell growth rate, capacity of colony formation and sensitivity to
genotoxic agent and cytolytic effect of parovirus H-1. Reduced growth
rate, capacity of colony formation and cytolytic effect to parvovirus
H-1 were observed and increased sensitivity to genotoxic agents. All
these effects were independent on number of integration DNA, furthermore
the integration was preferentially located on chromosome 17 not 19.
However, some other integrations may cause cell transformation as
reported that hepatocaricnoma causing integration was located in
chromosome 12 [26]. Therefore integration is dual-edge sword and
specific integration site should be created and clarified. More further
investigation on AAV DNA genome integration is required for more secure
and stable transgene expression and satisfactory therapeutic effects.
Mechanism for DNA integration or recombination should be investigated
for this purpose as well.

Receptor-ligand interaction: All the AAV vectors contain viral
coat, which is capsid protein, therefore the most popular and profound
progress in virus mediated targeting is mainly about the viral capsid
modification. Receptor-ligand interaction mediated targeting gene
delivery relies on the well-known target molecule such as cancer
associated/related antigen. Computer technology based bio-informatics
analysis predicts the candidate peptide bound to target molecules. Phage
display libraries are applied for potential peptides screening binding
to known/unknown target molecules. Bacteriophage (phage) evolved as
bacterial viruses, but can be adapted to transduce mammalian cells
through ligand-directed targeting to a specific receptor. AAVP provides
superior tumor transduction over phage and that incorporation of
inverted terminal repeats is associated with improved fate of the
delivered transgene [36]. Peptide screening from phage peptide display
library is the major means to discover peptides bound to target
molecules. However, there are still some limitations restricting the
obtainment of high effective targeting peptide. New technology should be
explored for screening more specific and selective peptide for targeting
molecules. Capsid modification based receptor-ligand interaction
strategy is categorized into the following groups: (1) non- genetically
modified AAV capsid, such as bi-specific antibody conjugated to capsid
surface, (2) exogenous peptide genetic insertion modified capsid (mosaic
capsid), (3) serotype capsid mixture (chimeric serotype capsid) and (4)
serotype capsid and genome switch (transcapsidation).

Chemical conjugation modified viral capsid: Targeting-molecule
conjugate can directly modify virion binding with re-targeted cells. The
delivery specificity is based on the re-targeting molecule selectivity,
such as antibody, targeting peptide. Bispecific antibody labeled
adeno-associated virus exerted the alternative tropism and viral
infectivity and tumor selectivity are increased [47,70,11,114]. These
bispecific antibodies linked the virions together with the target cells
and the virions are engulfed into the targeted cells [47]. Yang
engineered a recombinant AAV virion carrying the chimeric capsid protein
with the variable region of a single-chain antibody against human CD34
molecules, a cell surface marker for hematopoietic stem/progenitor
cells. And this recombinant AAV virion achieved an increased
preferential infectivity in CD34+ human myoleukemia cell line KG-1,
which is normally refractory to rAAV transduction [121]. Carlisle
demonstrated that polymer-coating techniques could also be used to
modify and retarget adeno-associated virus (AAV) types 5 and 8 [12].
Efficient covalent attachment of poly-[N-(2-hydroxypropyl)
methacrylamide] (HPMA) copolymer to AAV5 could only be achieved
following modification of the virus with carbodiimide (EDC). Delivery of
AAV5 genomes using polyethylenimine (PEI) and HPMA was efficient and
provided absolute control of tropism and protection from antisera.
However, the coating efficiency is very critical for the satisfactory
targeting effect. Meanwhile the side effect of the coating agents such
as PEI should be considered.

Genetic modified viral capsid: Mosaic capsid modifications contain
the exogenous insertion introducing the possible interaction with target
cells via inserted peptides or large molecules
[10,19,32,34,70,87,96,106,114,115]. Among the all applied insertion
peptide and molecules, RGD4C is the most popular peptide for viral
capsid modification to target the intergrin on the cellular surface
majorly expressed on the tumor cells [32,34,54,87-90]. AAV mosaics
revealed the selective and efficient transduction in targeted tumor cell
[19,32,91]. The application of AAV2 mosaics with a protein A fragment
inserted into their capsid, together with targeting antibodies, is a
versatile method that allows the specific transduction of a wide array
of cell types[32]. VEGF peptide, EGF peptide, FGF peptide, LH peptide
have also been studied for their targeting function from different group
[6,23,72,87,99]. However, all these mosaic capsid and bi-specific
antibody modified capsid carry the native tropism and modification
provides the viral alternative interaction with target cells. To
precisely control and modulate the viral tropism, native tropism needs
to be abrogated and only novel tropism from modification will be
functional. Replicating adenovirus with mutated capsid proteins, in
which the promiscuous adenovirus native tropism was abolished and a
bi-specific adapter molecule to target the virus to the Epidermal Growth
Factor Receptor (EGFR) was encoded[11]. Adenoassociated virus 2 natural
tropism has been abrogated via double mutation in capsid at site 520 and
584 and a novel tropism was achieved via inserted RGD4C peptide proved
by in vitro RGD-integrin mediated specific infectivity and heparin
non-dependant infectivity [90]. Metabolically biotinylated AAV was
produced via biotin peptide inserted into AAV capsid gene, such as
serotype 1, 2, 3, 4 and 5. Due to high affinity and interaction between
biotin and avidin, this strategy not only provides the unique platform
for different serotype AAV purification, but also alters AAV tropism via
biotin-mediated interaction with avidin-engineered cellular receptor
[4]. Genetic modification on AAV vectors may overcome the barrier from
AAV neutralizing antibody in vivo evidenced by AAV mutant's reduced
affinity to neutralizing antibody [45], indicating the AAV mutants not
only introduce the selective transduction, but also make AAV
re-administration feasible and rational in gene therapy application.
Meheshri reported an approach involving the generation of large mutant
capsid libraries and selection of Adeno-Associated Virus (AAV) 2
variants with enhanced properties including altered tropism, evasion
from neutralizing antibody. This novel approach provides a high
throughput selection process and directs to generate the
"designer" gene delivery system with specific properties [60].
However, there are still some barriers of the genetic insertion
modification of viral capsid in the achievement of targeting gene
therapy, such as the specificity of inserted peptide, the titer of
modified AAV vector and peptide binding ability.

Mixed serotype viral capsid: Serotype swapping among AAVs can endue
different serotype AAV with new alternative tropism, which expanded the
viral tropism and enhance the AAV mediated gene transduction as well
[77]. Chimeric capsid contains the mixed capsid proteins from different
serotype AAV or adenovirus, which was made by complementation with
separate plasmids mixed at various ratios. During the vial assembly, the
capsid proteins from different serotype were theoretically packaged into
virion, depending on ratio of complementing plasmids. And moreover this
mixed serotype capsid plasmids are up to five serotype
[19,27,42,76,77,79,86]. Although the actual ratio of the each capsid
from different serotype is difficult to determine, the data achieved
strongly indicated that this strategy is very promising to generate
sophisticated virus with multiple properties of different serotypes.
Chimeric vector was produced by using the mixture of AAV2 and AAV1
capsid helper plasmids in the transfection process. Recombinant chimeric
vector carried both parent serotype characters, such as purification by
heparin sulfate column, gene transduction in both muscle (AAV1) and
liver (AAV2) [41]. Replacement of AAV2 capsid VP1 N terminus domain from
amino acid 350 to 430 with corresponding domain of AAV1 capsid VP1
allowed the hybrid vector formation. In vitro characterization analysis
of this hybrid vector showed that it had stronger heparin-sulfate
binding affinity, higher stability of viral particles and more efficient
gene transduction in muscle fiber cells, which indicated the hybrid
vector achieved both tropism from parent vectors and may facilitate the
initiation of clinical application study [43]. AAV vector achieved via
this mixed serotype capsid transfection strategy may be difficult to be
repeatable. Therefore it might not be ideal to develop this means for
gene therapy or gene transfer. However, this strategy might play
important role for viral structure studying and viral development
analyzing.

Transcapsidation of different serotype capsid and AAV genome: AAV
transcapsidation is a good method to study how the different serotype
affects the gene transfer, gene transduction and gene expression. AAV2
is the best characterized serotype and served as the archetype for AAV
replication study. Due to the well knowledge of genetic/biochemical
properties of AAV2 and the host cell response to the AAV2 ITR, AAV2ITR
was first cross-packaged into the serotype AAV4 capsid and compared the
transduction efficiency to AAV5 vector containing AAV5ITR genome. The
experiments was unsuccessful because the ITRs were from different
serotype [124]. More studies have been carried out by packaging AAV2ITR
genome with different serotype capsids and gene transduction efficiency
has been further analyzed. Through this strategy it was found that
efficient transduction mediated by AAV8 was due to rapid uncoating of
vector genome [3,46,101,113]. A drawback of this strategy is that the
transencapsidated AAV2 ITR into AAV5 capsid vector suffered from low
titer yield, because the trs of AAV5 is significantly different from the
trs of AAV1, 2, 3, 4 and 6 and Rep78/68 cannot efficiently nick or
process the AAV5 ITR trs [18]. A further complication is that while the
N-terminus of Rep mediates specific ITR binding, the C-terminus appears
to interact specifically with the capsid [50]. Therefore, an alternative
strategy is required for generating higher titer cross-packaged virus.
In order to study the role of serotype-specific shell on viral
transduction, a more comprehensive study was performed to crosspackage
AAV2 ITR flanked vector genome into the capsids of AAV1, 2, 3, 4 and 5
by engineering chimeric AAV genome containing different serotype capsid
gene and AAV2 replication gene [76,77]. Of the five serotype vectors,
only types 2 and 3 were efficiently purified by heparin-Sepharose column
chromatography, illustrating the high degree of similarity between these
virions. These data established a hierarchy for efficient
serotype-specific vector transduction depending on the target tissue.
These data also strongly support the need for extending these analyses
to additional animal models and human tissue. The development of these
helper plasmids should facilitate direct comparisons of serotypes, as
well as begin the standardization of production for further clinical
development. The efficiency of interaction between the hybrid Reps and
the serotype-specific capsids is increased, producing titers of
cross-packaged vectors similar to that of AAV2. Different serotype AAV
vectors carry different tropisms, therefore mixture of different
serotype AAV capsid protein may help providing the custom-designed AAV
vector. As shown by [76], different ratio of capsid mixture affected the
virions assembly, physical titer and infectivity. And this approach may
be of great significant value to the field of targeting gene therapy.

Neutralizing antibody may partially or completely block the vector
mediated gene transduction when the same serotype vector is
administrated. Therefore serotype swapping or cross-packaging capsid
protein have shown to solve this barrier at different level [37].

Taken together, all the currently available genetic modified AAV
targeting vectors need to be improved not only with regard to the
elimination of the wild-type AAV tropism and but also the enhancement of
viral assembly efficiency and selective infectivity. A better
understanding of trans-membrane internalization and intracellular
trafficking will provide the route to develop the more effective AAV
vectors. New methodologies made to tailor the tropism of AAV have
improved the transduction and selectivity profiles. Novel modification
strategies have resulted in unique AAV vectors characterized by unique
capsid protein sequences that employ alternative receptors.

Tissue-specific transcription: Tumor Specific Promoter (TSP) makes
it applicable to initiate the targeting expression of interested genes
in the specific tissues and cells [98]. Application of TSP is one of the
oldest means ever used for targeting gene therapy and still being used
widely. Human TERT is the most widely used tumor specific promoter.
There are more TSPs applied in the preclinical study and clinical study
as well, such as AFP [116], CEA [9], CXCR4 [54], surviving [29,127],
osteocalcin [38,62], RPE targeting promoter [98]. TSPs are more applied
in cancer gene therapy such as hTERT, AFP. AAV has been reported to
inhibit the promoter activity of some oncogene and viral gene including
human papillomavirus type 16 [25,93,122,125]. AAV Rep78 was confirmed to
inhibit transcription initiation of the HPV-16 LCR by disrupting the
interaction between TATA binding protein and the TATA box of the p97
core promoter [93,125]. AAV Rep 78, not Rep68, was shown to be able to
inhibit the CREB-dependent transcription pathway, indicating that AAV
may disturb the cyclic AMP response pathway in the viral infected cell.
Thereafter, it is suggested that this AAV Rep78 protein might be used to
treat for HPV-induced infection and cancer such as cervical cancer.
Cucchiarini demonstrated the selective transferred gene expression in
microglia and macrophage lineage mediated by AAV-derived vector
containing cell-type-specific transcriptional element regulating
interest gene expression [21]. Transcriptional targeting were achieved
via a novel AAV-based system in which rAAV vectors were generated
harboring a luciferase reporter gene under the control of 1.5-kb cardiac
myosin light chain promoter fused to the CMV immediate-early enhancer
(CMV(enh)/MLC1.5) [69]. Chen reported the specific anti-cancer effect
mediated by AAV2 vector encoding p53 gene driven by AFP promtoer in
hepatoma cells [17]. Hypoxia responsive promoter has been applied for
driving oncolytic virus replication according to the tumor hypoxia
environment [58,92,82]. The hypoxiaresponsive promoter 5HREp, in which
five copies of the Hypoxia-Response Element (HRE) enhance transcription,
was employed to induce the expression of BCD (bacterial cytosine
deaminase) under hypoxic conditions. However, cell-type-specific
promoter or regulation elements have not been applied or investigated in
AAV as widely as adenovirus. All these tissue specific expression
regulation elements are to be applied in AAV mediated gene therapy
studies as well. Low interested gene expression is the major barrier for
the targeting transcription. Then how to increase the gene expression is
an important issue for wide application of transcription targeting. Some
elements which are able to enhance the promoter initiated transcription
ability are under investigated as well. Inducible tissue specific
transcription cassette and powerful post-transcription regulatory
element have been reported and high selective and continuous gene
expression was maintained for a long period of time.

AAV engineered cytotherapy: AAV vector engineered cells also can be
applied for cytotherapy, such as hemotopoietc stem cells
[57,85,100,110,128]. HSCs are easily accessible and can be readily
delivered back to patients by autologous transplantation, which renders
them as attractive targets for ex vivo gene therapy. The AdenoAssociated
Virus (AAV) vectors have to date not been associated with any malignant
disease and have gained attention as a potentially safer alternative to
the more commonly used retroviral vectors for HSC gene therapy. There
exists a conflicting data with regard to HSC transduction by AAV
vectors. AAV transduced Dentritic Cells (DC) expressing cancer antigen
were potent activator of CD8+ T cells, indicating these AAV engineered
DC cells could be developed into a new DC-based tumor vaccine.
Self-complementary AAV1 or AAV2 were able to transduce ex vivo
conventional DC, LC, or pDC more efficiently than single strain AAV1 or
AAV2. These self-complementary AAV vector exerted direct targeting to DC
cells in vivo as well [105]. AAV2 transduced human keratinocytes in ex
vivo culture were applied to generate transgene-positive recombinant
skin (r-skin), using the organotypic epithelial raft culture system [1].

Present Obstacles for AAV vectors: Even though it has been widely
accepted that AAV vector is nonpathgenic, there are still some research
reports about the possible pathological alternations induced by this
vector. Schimidt reported that AAV2-Rep78 protein mediated apoptosis is
caspase-2 dependent in wild-type p53 and p53-dull human cells, the
possible mechanism of apoptosis in part is due to DNA binding and
ATPase/helicase activity of Rep 78 protein, not its endonuclease
activity [83]. Human cytomegalovirus (HCMV) is considered as a competent
helper for complete replication of AAV, similar with adenovirus and
herpes simplex virus. Accumulation of AAV capsid antigen and infectious
AAV were observed in fibroblast cells co-infected with CMV and AAV and
there was a 24 h lag of AAV replication compared to AAVadenovirus
co-infection. Furthermore the synergistic cytopathic effect was induced
as well, indicating AAV contributing to CMV-induced pathogenicity [64].
Recent studies illustrated that both normal mice and mice with
mucopolysaccharidosis VII (MPS VII) developed hepatocellular carcinoma
(HCC) after neonatal injection of an AAV vector expressing
b-glucuronidase. These four tumors were confirmed to carry the
integrated AAV DNA located within a 6-kilobase region of chromosome 12.
Random integration of AAV vector causing untoward event raises the
concerns of further clinical application of AAV vectors and requires the
more clear specific integration site.

Future of AAV vectors: Targeting gene therapy has been studied
broadly and widely. Modifications of different gene therapy vector have
been made and targeting gene delivery also has been obtained with a
varied specificity. However, the present receptor-ligand interaction
mediated targeting is mainly dependent on the known target molecules.
Unfortunately there is no absolute tumor associated antigen found so
far, which may limit the tumor target effectiveness. Meanwhile, peptides
or protein molecules desired for tumor associated antigen targeting are
obtained from phage display screening and computer analysis. The phage
enrichment process affects the affinity screening results and meanwhile
the same sequence peptide expressed on gene therapy vector might not
bind to target protein with the same affinity as it is expressed on
phage. Therefore some new technologies need to be developed to discover
the high affinity peptide to the already know target molecules and
potential unknown tumor related antigens. Using the character of wild
type virus infectivity and replication in host cells, the tumor cell
specific virus can be discovered by its natural tropism and replication.
This technology not only provides the high selective ligand binding to
the tumor cells but also provide the possibility to discover the new
tumor associated antigens. The alternative tropism can be introduced for
chimeric AAV via AAV serotype swapping. Not only these hybrid serotypes
could achieve high efficiency of gene delivery to a specific targeted
cell type, which can be better-tailored for a particular clinical
application, but also serve as a tool for studying AAV biology such as
receptor binding, trafficking and genome delivery into the nucleus.
Along with the broader clinical trials, further basic virological
investigations and immune response involvement, targeting gene transfer
and targeting DNA integration are very critical for safe issues and
better therapeutic effects. Site specific integration of AAV is the next
generation vector development direction. And high throughput and fast
selection process for peptide screening for directing to the target
molecules and to discover more target antigens as well is another
possible trend for AAV targeting gene therapy.

ACKNOWLEDGEMENT

We thank Dr. Zhang C. at University of Florida and Dr. Zhou
Xiangjun at Shanghai Jiaotong Unvieristy for the improvement of English
writing. The study was supported by Nature and Science Foundation of
China (30772477).