Biotechnol Lett (2009) 31:321–328
DOI 10.1007/s10529-008-9869-0
REVIEW
The treatment of hemophilia A: from protein replacement
to AAV-mediated gene therapy
Shen Youjin Æ Yin Jun
Received: 18 July 2008 / Revised: 4 October 2008 / Accepted: 7 October 2008 / Published online: 2 November 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Factor VIII (FVIII) is an essential
component in blood coagulation, a deficiency of
which causes the serious bleeding disorder hemophilia A. Recently, with the development of
purification level and recombinant techniques, protein replacement treatment to hemophiliacs is
relatively safe and can prolong their life expectancy.
However, because of the possibility of unknown
contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use
these products, gene therapy for hemophilia A is an
attractive alternative to protein replacement therapy.
Thus far, the adeno-associated virus (AAV) is a
promising vector for gene therapy. Further improvement of the virus for clinical application depends on
better understanding of the molecular structure and
fate of the vector genome. It is likely that hemophilia
will be the first genetic disease to be cured by somatic
cell gene therapy.
Keywords Adeno-associated virus Factor VIII Gene therapy Hemophilia A
S. Youjin (&)
Department of Hematology, The Second Hospital
of Shantou University Medical College,
515041 Shantou, China
e-mail: [email protected]
Y. Jun
Department of Laboratory, The Second Hospital
of Shantou University Medical College,
515041 Shantou, China
Introduction
Hemophilia A is an X chromosome-linked recessive
bleeding disorder caused by mutations in the gene for
factor FVIII (FVIII), termed hemophilia A (High
2002). FVIII is involved in the intrinsic pathway of
blood coagulation (Fig. 1) (Fricker 2001), and affected
individuals can have severe, moderate or mild forms
of the disease, which are defined by Factor plasma
levels of \1.1%, 1.1–5%, and [5% of normal levels,
respectively (Mannucci and Tuddenham 2001).
Clinically, the disease is characterized by frequent,
spontaneous bleeding episodes, mostly into the joints
and soft tissues, easy bruising, and prolonged bleeding
(High 2002; Hauck et al. 2006). The preference for
bleeds into joints is still not well understood (High
2002). Recurrent bleeds into the joints, primarily the
knees, ankles, and elbows, constitute the major morbidity of the disease, and eventually result in an
arthropathy that limits rang of motion in the joints.
Bleeding can also occur into other critical closed
spaces, such as the intracranial or the retroperitoneal
space, where it can be severe and can lead to death
(High 2002, Hauck et al. 2006). Generally, because of
its X-linked and hereditary, men are affected and
women are carriers. However, there are few reports
about acquired hemophilia A, which is caused by
suddenly appearing autoantibodies that interfere
with coagulation FVIII activity (Flisiński et al.
2008; Collins et al. 2008; Akahoshi et al. 2008;
Alvarado et al. 2007; Mahipal and Bilgrami 2007).
123
322
Biotechnol Lett (2009) 31:321–328
Fig. 1 The coagulation
cascade, which leads to the
conversion of fibrinogen to
insoluble fibrin
The prevalence of the disease is evenly distributed
worldwide. Hemophilia affects 1 in 5,000 male in the
USA, with hemophilia A being the more prevalent
treatment form (approximately 2/3 of patients) (Wang
and Herzog 2005). The World Federation of Hemophilia estimates that of the 400,000 individuals
worldwide with hemophilia, 300,000 receive either
no, or very sporadic, treatment. Therefore, it is urgent
to find a economic and convenient way to cure
hemophilia. Gene therapy is an attractive way to
completely cure this hemophilia compared to traditionally infusion of coagulation factor protein. In the
following, progress and problem on AAV-mediated
gene therapy for hemophilia A is summarized.
Current protein-based therapy and gene therapy
Current treatment of hemophilia is based on the
episodic intravenous infusion of highly purified
plasma-derived or recombinant clotting factor concentrates, mostly in response to bleeding episodes
(on-demand therapy) (Larson and High 2001). The
widespread introduction of this form of therapy in
the 1970s resulted in a dramatic improvement in life
expectancy for people with hemophilia (High 2002).
Although the plasma-derived products are now
generally considered safe, effective, and compatible
with a normal lifespan, in the past contamination with
viruses, including hepatitis A, B and C, and HIV, has
123
occurred (Ragni 2004). Recently, new concerns
regarding contamination by the putative cause of
variant Cruetzfeldt-Jakob disease, prion proteins,
have been raised (Couto and Pierce 2003). The
cloning of the FVIII gene and the production of
recombinant proteins has solved some of these
problems. However, these proteins are difficult and
therefore costly to produce (costs that can reach US
$100,000 to $200,000 per year for an individual with
severe hemophilia) and periodic product shortages
(Couto and Pierce 2003). Although a protein replacement approach is effective in many patients with mild
and moderate hemophilia, it is limited by several
factors including the short in vivo half-life, the high
cost and low availability of purified FVIII. In
addition, infusion therapy is also inconvenient in that
it requires gaining intravenous access (Larson and
High 2001). Because of the expense of frequent
factor infusions, treatment is rarely prophylactic,
with the result that bleeding is treated rather than
prevented (Bristol et al. 2001). Consequently,
repeated damage to joints takes place, and the risk
of a fatal bleed remains (Bristol et al. 2001).
These unmet needs have fuelled interest in gene
therapy of hemophilia A because of its potential to
ameliorate or cure the bleeding diathesis following a
single maneuver that mediates persistent therapeutic
expression of implicated coagulation protein. Hemophilia A is an excellent candidate for gene therapy for
the following reasons. Firstly, it is a single-gene
Biotechnol Lett (2009) 31:321–328
disorder and FVIII expression does not appear to be
subjected to tight regulation (Larson and High 2001;
Garcı́a-Martı́n et al. 2002). The data from primary
prophylaxis studies imply that levels as low as 1–2%
may be beneficial in preventing complications of the
disease (Larson and High 2001). In addition, experience with the use of highly purified coagulation
factor concentrates suggest that if gene transfer were
so efficient as to result in plasma levels of factor of
over 100%, such levels would not result in thrombosis (Larson and High 2001). Secondly, tissue specific
expression of coagulation FVIII is not a prerequisite
(Garcı́a-Martı́n et al. 2002). As long as the coagulation protein expressed in a particular tissue undergoes
the critical post-translational processing events that
lead to activity and gain access to the circulation, any
tissue type would be a potential target for gene
transfer (High 2002; Larson and High 2001). Thirdly,
many well characterized animal models exist for
hemophilia A, such as naturally occurring dog
models (Evans et al. 1989; Cameron et al. 1998)
and the engineered FVIII gene knock-out mice (Bi
et al. 1995; Lin et al. 1997). Finally, determination of
therapeutic effect is straightforward since the APTT
and clotting factor activity levels can be readily
measured in most laboratories (Larson and High
2001).
Vectorology for hemophilia A gene therapy
Over the past ten years, significant advances have
been made in the development of novel vector
systems for the treatment of hemophilia A by gene
therapy. A widely pursued strategy for treatment is
in vivo gene transfer using viral or non-viral vector
systems for expression of functional coagulation
factor in a specific target tissue (Wang and Herzog
2005). Using the viral vectors predominates in
hemophilia A gene therapy due to a higher efficiency
of gene transfer and sustained long-term transgene
expression (Chao and Walsh 2002). Thus far, viral
vectors are generated from the retrovirus Moloney
murine leukemia virus (MMLV), herpes simplex
virus (HSV), adenovirus (Ad), adeno-associated virus
(AAV) and lentivirus (Chao and Walsh 2002;
Mori et al. 2008; Vandendriessche et al. 2007;
Brunetti-Pierri et al. 2005; Brown et al. 2007).
In the case of hemophilia A, early work with
323
retroviral, lentiviral and adenoviral vectors have been
disappointing, since these vectors were not able to
achieve sustained expression and achieve protein
expression at levels high enough to have a therapeutic
effect (High 2001). In contrast, AAV vectors have
become favorites in many laboratories because of
efficient in vivo gene transfer to non-dividing targets
cells, the ability to direct sustained expression
(several years in canine models), and reduced
immunogenicity compared to other vector systems
(Wang and Herzog 2005). In the following, we will
emphasize on hemophilia A gene therapy using
recombinant adeno-associated viral vectors (rAAVs).
AAV is a small, single-stranded DNA virus with a
genome of approx. 4.86 kb and is a member of the
parvovirus family. It is a non-pathogenic, replicationdefective human virus that has been developed into a
gene-delivery vector due to its high efficiency of
infection for many different cell types and its ability to
persist and lead to long-term gene expression (Choi
et al. 2007). Pseudotyping studies show that the
differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in
their capsid viral protein (VP) amino acids (Nam et al.
2007). Towards identifying the structural features
underpinning these disparities, Nam et al. (2007)
determined the crystal structure of the AAV8 viral
capsid to 2.6 Å resolution. Also, the atomic structure
of AAV-2 has been determined to 3-Å resolution by
X-ray crystallography by Xie et al. (2002).
Of the twelve natural serotypes of AAV, serotype
2 (AAV-2), the first AAV serotype to be cloned and
sequenced, is the best characterized and has been the
most widely used in the majority of AAV gene
delivery study (Hauck et al. 2006; Chao and Walsh
2002; Mori et al. 2008). rAAV production and
purification techniques are primarily derived from
studies on AAV-2, which is the serotype used in the
majority of gene studies that utilize AAV (Samulski
et al. 1999; Li and Samulski 2005).
AAV-2 is a small (*20 nm diameter) DNA virus
containing a 4679 bp linear, single-stranded genome
(Chao and Walsh 2002; Ruffing et al. 1994). AAV-2
has a non-enveloped icosahedral capsid that encloses
the ssDNA genome containing two large openreading frames, rep and cap (Bleker et al. 2006).
The non-structural rep genes encode four proteins
designated Rep78, Rep68, Rep52 and Rep40 (Chao
and Walsh 2002). These proteins are required for
123
324
DNA replication, DNA encapsidation, and control of
gene expression (Bleker et al. 2006). The structural
cap genes encode the three capsid proteins VP1,
VP2 and VP3, of which protein VP3 is the most
abundant and accounts for 80% of the total virion
capsid (Chao and Walsh 2002). The VPs share a
common central and C-terminal region with
N-terminal amino acids forming the unique portion
of VP1 (Bleker et al. 2006). The only cis components
required to generate rAAV-2 vectors are the two
145-nucleotide inverted terminal repeats (Samulski
et al. 1999). These terminal repeats are required for the
replication and packaging of the recombinant genome
into the newly formed AAV virions (Samulski et al.
1999). To accommodate the gene expression cassette,
the AAV coding region is removed and only two
flanking cis elements, inverted terminal repeats (ITRs,
145 bp each), are preserved (Hauck et al. 2006). As
members of dependovirus genus of the parvoviridae
family, AAV replication is not autonomous and is relies
on functions of the gene products of helper viruses, such
as adenovirus,cytomegalovirus, Epstein-Barr virus,
varicella virus, pseudorabies virus, vaccinia virus,
herpes simplex virus or human papillomavirus (HPV),
in order to complete their lytic life cycle (Bleker et al.
2006; Murphy and High 2008; Ye and Pintel 2008;
Bandyopadhyay et al. 2008).
rAAV has the ability to transduce terminallydifferentiated, dividing and non-dividing cells, and
establishes latency as an episome (Chao and Walsh
2002; Hallek et al. 1998). Although the natural route
of infection for wild-type AAV is through the upper
respiratory tract, rAAV has demonstrated efficient
infection and long-term expression of transgenes in
most tissues, including the brain, liver, muscle, retina
and vasculature of experimental animals (Samulski
et al. 1999).
As AAV-based vectors approach the clinic, the
need for scalable methods of production and purification is steadily increasing (Smith et al. 2008).
Recent advances in the production and purification of
rAAV-2 vectors include the elimination of helper
virus (adenovirus) contamination by the use of
complementing Ad plasmids, and purification by
heparin affinity column chromatography (Samulski
et al. 1999). Smith et al. (2008) presented a column
chromatography-based protocol for the purification of
rAAV. The protocol, which can be completed within
one working day, employs three major purification
123
Biotechnol Lett (2009) 31:321–328
steps: (1) polyethylene glycol-mediated vector
precipitation, (2) anion-exchange chromatography,
and (3) gel filtration chromatography. Chen (2008)
successfully produced sufficient AAV vectors in
insect cells and Durocher et al. (2007) described the
production of rAAV-2 using suspension-grown
human embryonic kidney (HEK293) cells in serumfree medium. The AAV purification and production
methods have paved the way of an effective strategy
of achieving sufficient vectors for gene delivery.
AVV serotypes as gene transfer vectors
Since the isolation of AAV-2, many different AAV
serotypes have been isolated from human and
non-human primate tissues. Currently, twelve AAV
serotypes of various origin have been identified and
cloned (Chao and Walsh 2002; Mori et al. 2008;
Schmidt et al. 2008). Figure 2 (Schmidt et al. 2008)
shows the evolutionary relationship among human and
nonhuman primate AAVs. They are termed as serotype
1 to 12 in accordance with their unique serological
(immunological) characteristics, and the timing of their
isolation and characterization (Chao and Walsh 2002).
Seroepidemiological surveys revealed that AAV-1,
AAV-2, AAV-3, AAV-5 and AAV-6 infect human.
Otherwise, AAV-4 derived from African green monkey
cells rarely infects human, although it can infect human
cells in vitro (Chiorini et al. 1997). Generally, all
primate AAV show more than 80% homology in
Fig. 2 Evolutionary relationship among human and nonhuman
primate AAVs. The unrooted phylogenetic tree is based on
merged ClustalW alignments of partial genome sequences and
shows the relatedness of different AAVs; the lengths of the
branches are proportional to the evolutionary distances
between isolates
Biotechnol Lett (2009) 31:321–328
nucleotide sequence (Xiao et al. 1999). AAV-6 appears
to be a hybrid of AAV-1 and AAV-2 formed by
homologous recombination between highly conserved
regions spanning 452–552 nucleotides (Xiao et al.
1999). Vectors formed with capsids from AAV7 and
AAV8 were generated by using rep and inverted
terminal repeats (ITRs) from AAV2 and were compared
with similarly constructed vectors made from capsids of
AAV1, AAV2, and AAV5 (Gao et al. 2002).
AAV serotypes differ broadly in transduction
efficacies and tissue tropisms and thus hold enormous
potential as vectors for human gene therapy. In
comparison with the prototypic AAV2, AAV vectors
pseudotyped with other serotypes show superior
transduction efficiency in various tissues: AAV1 in
muscle, pancreatic islets, heart, vascular endothelium,
brain, central nervous system (CNS) and liver; AAV3
in Cochlear inner hair cells; AAV4 in brain, lung,
kidney, heart; AAV5 in brain and CNS, lung, eye,
arthritic joints, and liver; AAV6 in heart, liver,
skeletal muscle and airway epithelium; AAV7 in
muscle, liver; AAV8 in muscle, pancreas, heart, and
liver (Jiang et al. 2006); AAV9 in heart, liver, CNS
(Fechner et al. 2008; Zincarelli et al. 2008; Miyagi
et al. 2008; Klein et al. 2008); AAV10 in CNS,
hematopoietic stem cell (HSC), immune cells, skeletal muscle (Klein et al. 2008; Maina et al. 2008;
Mori et al. 2004, 2008); AAV11 in immune cells,
skeletal muscle (Mori et al. 2004, 2008); AAV12 in
HSC, muscle and salivary glands (Schmidt et al.
2008; Srivastava 2008).
AAV-mediated factor VIII gene transfer
The prevalence of hemophilia A is approximately six
times more prevalent than hemophilia B (Kaufman
1999), so it is quite urgent to find a good way to cure
hemophilia A. The gene encoding FVIII is made up
of 26 exons and is at 7.3 kb, which is much larger
than FIX. The treatment of hemophilia A by gene
transfer has lagged behind that of hemophilia B
because FVIII is inefficiently expressed (Couto and
Pierce 2003). Due to its large molecular weight and
the need for stabilization with von Willebrand factor,
FVIII transgene expression has largely been pursued
in the context of hepatic gene transfer (Wang
and Herzog 2005). Although circulating at low
concentration in human (normal plasma levels are
325
100–200 ng/ml instead of 5 ug/ml in the case of
FIX), FVIII has been more difficult to express at
therapeutic levels (Wang and Herzog 2005).
The gene encoding FVIII is 186 kb and the FVIII
amino acid sequence deduced from the cloned cDNA
identified a domain structure of A1-A2-B-A3-C1-C2.
Recombinant AAV vectors are limited in terms of the
size of the transgene they can package, and AAV
packaging and efficiency of transgene expression
decreases dramatically when the rAAV/transgene
expression cassette reaches 25 kb (Chao and Walsh
2002). Therefore, the obstacle is amplified by the
large size of the cDNA, and constrained by limited
packaging capacity, even the 4.3 kb B domaindeleted FVIII remained a challenge for delivery by
a single AAV vector (Lu et al. 2008). A vector
sequence up to 6.6 kb may be packaged into AAV
virions, which suggested an alternative strategy for
hemophilia A gene therapy (Lu et al. 2008). Furthermore, clinical use of recombinant B domain-deleted
(BDD) FVIII maintains biological activity and has
not shown an increased risk of inhibitor formation
compared to products based on the full-length
molecule (Wang and Herzog 2005).
Sarkar et al. (2003) reported partial correction of
hemophilia A mice using a single AAV-2 vector
expressing B domain-deleted murine FVIII. They
found that despite long-term phenotypic correction,
plasma FVIII activity peaked at only 8% with
intraportal administration and declined to 2% to 3%
at 9 months, and they attributed these modest levels
to the use of a short promoter lacking regulatory
elements necessary for greater FVIII expression and
poor transduction efficiency of the AAV-2 serotype.
Additionally, after evaluating AAV serotypes 2, 5, 7,
and 8 in gene therapy of FVIII deficiency in a
hemophilia A mouse model, they found that AAV-8
was superior to the other 3 serotypes and AAV-8
gave 100% correction of plasma FVIII activity
irrespective of the vector type or route of administration (Sarkar et al. 2004). Jiang et al (2006)
demonstrated short-term activity of a liver-specific
AAV-2 vector expressing canine B-domain-deleted
FVIII in a hemophilia canine model. After that, they
also reported the long-term (more than 3 years)
efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and
dogs (Jiang et al. 2006). Generally, high vector doses
(AAV-2 serotype) of 4 9 1012 – 2 9 1013 vg/kg
123
326
were required to obtain FVIII activity levels of 2–4%
of normal in hemophilia A mice and dogs.
Despite the improvement, the limited rAAV
packaging capacity still restricts the use of larger
regulatory elements that may improve FVIII expression (Chao and Walsh 2002). Recently, several dual
vector strategies have been developed to overcome
AAV packing constraints (Wang and Herzog 2005).
At first, it takes advantage of the molecular biology of
the AAV vector genome, which can form concatemers through intermolecular recombination (Nakai
et al. 2000). Thus far, there are no encouraging
reports to attain sufficient efficacy in gene
transduction.
The second strategy toward FVIII expression is
based on trans-splicing. Chao et al. (2003) carried out
splicesome-mediated RNA trans-splicing to repair
mutant FVIII mRNA, and as a result, a pre-transsplicing molecule corrected endogenous FVIII
mRNA in FVIII knockout mice with the hemophilia
A phenotype, producing sufficient functional FVIII to
correct the hemophilia A phenotype (Chao et al.
2003). FVIII is secreted as a heterodimer consisting
of a heavy chain and a light chain, which can be
expressed independently and reassociate with recovery of biological activity. Therefore, the third dual
vector strategy, which has been quite successful at
least in hemophilia A mice, is the expression of heavy
chain (Al, A2, and A3 domains) and light chain (Cl
and C2 domains) of FVIII from 2 separate vectors
(Wang and Herzog 2005). However, the FVIII heavy
chain is secreted 10–100 fold less efficiently than the
light chain. Chen et al. (2007) reported that in vitro
ligation of the light chain to the heavy chain
significantly increased heavy secretion, and such
heavy chain secretion increases were also confirmed
in vivo by hydrodynamic injection of FVIII intein
plasmids into hemophilia A mice. Moreover, similar
enhancement of heavy chain secretion can also be
observed when the light chain is supplied in trans. In
addition, since the majority of cells are transduced
with only one vector encoding either the heavy or
light chain, this strategy limits the amount of
functional FVIII that can be produced (Chao and
Walsh 2002). Using separate AAV-2 vectors to
deliver the heavy and light chains of FVIII, Scallan
et al. (2003) overcame the packaging limitations of
AAV, achieving phenotypic correction of hemophilia A
in mice.
123
Biotechnol Lett (2009) 31:321–328
Kasuda et al. (2008) found that embryoid bodies
developed under conditions promoting liver differentiation efficiently secreted human FVIII after
doxycycline induction. Moreover, use of a B-domain
variant FVIII cDNA (226aa/N6) dramatically
enhanced FVIII secretion. These findings suggest
the potential for future development of an effective
embryoid stem cell-based approach to treating hemophilia A. Using AAV-8, Sarkar et al. (2004) obtained
100% correction of FVIII deficiency regardless of the
vector type or route of administration. Rodriguez
et al. (2004) placed FVIII transgenes under the
control of the human platelet glycoprotein IIb (GPIIb)
promoter for stable transfection of the Dami megakaryocytic cell line and achieved high FVIII
production which had biological activity. These
results represent the first biochemical characterization
of megakaryocyte-produced FVIII.
The recent advances in gene transfer technology
have expedited the development of gene therapy for
the treatment of hemophilia A. Long-term therapeutic
levels of FVIII can now be achieved either in small
(mouse, rat) or large preclinical models (dog, nonhuman primate). Some of these encouraging preclinical studies culminated in several phase 1 clinical
trials for hemophilia A.
Other obstacle using AAV-mediated gene
therapy for hemophilia A
Like Factor IX gene transfer, there are additional
work is needed to identify which delivery method is
best, such as muscle and liver. Also, safety is the first
consideration and the effect of pre-existing immunity
to AAV on gene transfer and expression must be
evaluated through a number of experiments. Otherwise, the prevalence of clinically significant
inhibitors and potential of germline transmission of
vector need further experiments to assess.
Conclusions
The rate of development of therapeutic regimens for
the hemophiliac has dramatically escalated as our
knowledge about the structure, function, and mechanism of action of coagulation factors has become
more sophisticated. The hemophilias are considered
Biotechnol Lett (2009) 31:321–328
prime disorders for successful treatment using gene
therapy. Of the current viral vectors used in gene
transfer, rAAV is considered one of the most efficient
vectors for hemophilia gene therapy (Chao and Walsh
2002). The ability of the vector to efficiently transfer
genes to target organs in vivo, such as skeletal muscle
and liver, and to direct sustained expression has been
illustrated in large animal and clinical studies.
Successes in murine and canine models of hemophilia
A have been spectacular, resulting in long-term
correction of the bleeding disorder (Wang and
Herzog 2005). To date there has been no direct
evidence that AAV vector integrates into genomic
DNA in skeletal muscle and germline tissue (Larson
and High 2001). Some reports demonstrated stable
therapeutic FVIII levels in rabbits and partial correction of canine hemophilia A through a combination of
improved vector design, the ability to generate high
titers and better expression cassettes. However, there
are still some problems to be solved in the future
studies. In terms of pre-clinical research, efficient
AAV-mediated FVIII gene transfer, vector/host interactions which encompass immune responses, genome
integration and vertical germline transmission still
has to be overcome in a large animal model (Wang
and Herzog 2005). Thus, continued animal experiments and clinical research on innovative strategies
to cure hemophilia A are essential to continue to
build upon the results to date.
Acknowledgments This work was supported by Natural
Science Fund of Guangdong.
References
Akahoshi M, Aizawa K, Nagano S et al (2008) Acquired
hemophilia in a patient with systemic lupus erythematosus:
a case report and literature review. Mod Rheumatol Jun 13
Alvarado Y, Yao X, Jumper C et al (2007) Acquired hemophilia: a case report of 2 patients with acquired factor VIII
inhibitor treated with Rituximab plus a short course of
steroid and review of the literature. Clin Appl Thromb
Hemost 13:443–448
Bandyopadhyay S, Raney KD, Liu Y et al (2008) AAV-2
Rep78 and HPV-16 E1 interact in vitro, modulating their
ATPase activity. Biochemistry 47:845–856
Bi L, Lawler AM, Antonarakis SE et al (1995) Targeted disruption of the mouse factor VIII gene produces a model of
haemophilia A. Nat Genet 10:119–121
Bleker S, Pawlita M, Kleinschmidt JA (2006) Impact of capsid
conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging. J Virol 80: 810–820
327
Bristol JA, Gallo-Penn A, Andrews J et al (2001) Adenovirusmediated Factor VIII gene expression results in attenuated
anti-Factor VIII-specific immunity in hemophilia A mice
compared with Factor VIII protein infusion. Hum Gene
Ther 12:1651–1661
Brown BD, Cantore A, Annoni A et al (2007) A microRNAregulated lentiviral vector mediates stable correction of
hemophilia B mice. Blood 110:4144–4152
Brunetti-Pierri N, Nichols TC, McCorquodale S et al (2005)
Sustained phenotypic correction of canine hemophilia B
after systemic administration of helper-dependent adenoviral vector. Hum Gene Ther 16:811–820
Cameron C, Notley C, Hoyle S et al (1998) The canine factor
VIII cDNA and 50 -flanking sequence. Thromb Haemost
79:317–322
Chao H, Walsh CE (2002) Hemophilia gene therapy: novel
rAAV vectors and RNA repair strategy. Curr Opin Mol
Ther 4:499–504
Chao H, Mansfield SG, Bartel RC et al (2003) Phenotype
correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing. Nat Med 9:1015–1019
Chen H (2008) Intron splicing-mediated expression of AAV
Rep and Cap genes and production of AAV vectors in
insect cells. Mol Ther 16:924–930
Chen L, Zhu F, Li J et al (2007) The enhancing effects of the
light chain on heavy chain secretion in split delivery of
factor VIII gene. Mol Ther 15:1856–1862
Chiorini JA, Yang L, Liu Y et al (1997) Cloning of adenoassociated virus type 4 (AAV4) and generation of
recombinant AAV4 particles. J Virol 71:6823–6833
Choi VW, Asokan A, Haberman RA et al (2007) Production of
recombinant adeno-associated viral vectors. Curr Protoc
Hum Genet Chapter 12:Unit 12.9
Collins P, Budde U, Rand JH et al (2008) Epidemiology and
general guidelines of the management of acquired haemophilia and von Willebrand syndrome. Haemophilia
14:49–55
Couto LB, Pierce GF (2003) AAV-mediated gene therapy for
hemophilia. Curr Opin Mol Ther 5:517–523
Durocher Y, Pham PL, St-Laurent G et al (2007) Scalable
serum-free production of recombinant adeno-associated
virus type 2 by transfection of 293 suspension cells.
J Virol Methods 144:32–40
Evans JP, Brinkhous KM, Brayer GD et al (1989) Canine
hemophilia B resulting from a point mutation with unusual consequences. Proc Natl Acad Sci 86:10095–10099
Fechner H, Sipo I, Westermann D et al (2008) Cardiac-targeted
RNA interference mediated by an AAV9 vector improves
cardiac function in coxsackievirus B3 cardiomyopathy.
J Mol Med Jun 12
Flisiński M, Windyga J, Stefańska E et al (2008) Acquired
hemophilia: a case report. Pol Arch Med Wewn 118:228–233
Fricker J (2001) Viral gene therapy for haemophilia. Drug
Discov Today 6:165–166
Gao GP, Alvira MR, Wang L et al (2002) Novel adeno-associated
viruses from rhesus monkeys as vectors for human gene
therapy. Proc Natl Acad Sci 99:11854–11859
Garcı́a-Martı́n C, Chuah MK, Van Damme A et al (2002)
Therapeutic levels of human factor VIII in mice implanted
with encapsulated cells: potential for gene therapy of
haemophilia A. J Gene Med 4:215–223
123
328
Hallek M, Girod A, Braun Falco M et al (1998) Recombinant
adeno-associated virus vectors. IDrugs 1:561–573
Hauck B, Xu RR, Xie J et al (2006) Efficient AAV1-AAV2
hybrid vector for gene therapy of hemophilia. Hum Gene
Ther 17:46–54
High KA (2001) AAV-mediated gene transfer for hemophilia.
Ann N Y Acad Sci 953:64–74
High K (2002) AAV-mediated gene transfer for hemophilia.
Genet Med 4:56–61
Jiang H, Lillicrap D, Patarroyo-White S et al (2006) Multiyear
therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs. Blood
108:107–115
Kasuda S, Kubo A, Sakurai Y et al (2008) Establishment of
embryonic stem cells secreting human factor VIII for cellbased treatment of hemophilia A. J Thromb Haemost May 15
Kaufman RJ (1999) Advances toward gene therapy for hemophilia at the millennium. Hum Gene Ther 10:2091–2107
Klein RL, Dayton RD, Tatom JB et al (2008) Tau expression
levels from various adeno-associated virus vector serotypes produce graded neurodegenerative disease states.
Eur J NeuroSci 27:1615–1625
Larson PJ, High KA (2001) Gene therapy for hemophilia B:
AAV-mediated transfer of the gene for coagulation factor
IX to human muscle. Adv Exp Med Biol 489:45–57
Li C, Samulski RJ (2005) Serotype-specific replicating AAV
helper constructs increase recombinant AAV type 2 vector
production. Virology 335:10–21
Lin HF, Maeda N, Smithies O et al (1997) A coagulation factor
IX-deficient mouse model for human hemophilia B. Blood
90:3962–3966
Lu H, Chen L, Wang J et al (2008) Complete correction of
hemophilia A with adeno-associated viral vectors containing a full-size expression cassette. Hum Gene Ther
19:648–654
Mahipal A, Bilgrami S (2007) Acquired hemophilia in chronic
lymphocytic leukemia. Leuk Lymphoma 48:1026–1028
Maina N, Han Z, Li X et al (2008) Recombinant self-complementary adeno-associated virus serotype vector-mediated
hematopoietic stem cell transduction and lineage-restricted,
long-term transgene expression in a murine serial bone
marrow transplantation model. Hum Gene Ther 19:376–383
Mannucci PM, Tuddenham EG (2001) The hemophilias: from
royal genes to gene therapy. N Engl J Med 344:1773–
1779
Miyagi N, Rao VP, Ricci D et al (2008) Efficient and durable
gene transfer to transplanted heart using adeno-associated
virus 9 vector. J Heart Lung Transplant 27:554–560
Mori S, Wang L, Takeuchi T et al (2004) Two novel adenoassociated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein. Virology 330:375–
383
Mori S, Takeuchi T, Enomoto Y et al (2008) Tissue distribution of cynomolgus adeno-associated viruses AAV10,
AAV11, and AAVcy.7 in naturally infected monkeys.
Arch Virol 153:375–380
Murphy SL, High KA (2008) Gene therapy for haemophilia. Br
J Haematol 140:479–487
Nakai H, Storm TA, Kay MA et al (2000) Recruitment of
single-stranded recombinant adeno-associated virus
123
Biotechnol Lett (2009) 31:321–328
vector genomes and intermolecular recombination are
responsible for stable transduction of liver in vivo. J Virol
74:9451–9463
Nam HJ, Lane MD, Padron E et al (2007) Structure of adenoassociated virus serotype 8, a gene therapy vector. J Virol
81:12260–12271
Ragni MV (2004) Hemophilia gene transfer: comparison with
conventional protein replacement therapy. Semin Thromb
Hemost 30:239–247
Rodriguez MH, Plantier JL, Enjolras N et al (2004) Biosynthesis of FVIII in megakaryocytic cells: improved
production and biochemical characterization. Br J Haematol 127:568–575
Ruffing M, Heid H, Kleinschmidt JA (1994) Mutations in the
carboxy terminus of adeno-associated virus 2 capsid
proteins affect viral infectivity: lack of an RGD integrinbinding motif. J Gen Virol 75:3385–3392
Samulski R, Sally M, Muzyczka N (1999) Adeno-associated
viral vectors. In: Friedmann T (ed) The development of
human gene therapy. Cold Spring Harbor Laboratory
Press, New York, pp 131–172
Sarkar R, Xiao W, Kazazian HH Jr et al (2003) A single
adenoassociated virus (AAV)- murine factor FVIII. J
Thromb Haemost 1:220–226
Sarkar R, Tetreault R, Gao G et al (2004) Total correction of
hemophilia A mice with canine FVIII using an AAV 8
serotype. Blood 103:1253–1260
Scallan CD, Liu T, Parker AE et al (2003) Phenotypic correction
of a mouse model of hemophilia A using AAV2 vectors
encoding the heavy and light chains of FVIII. Blood 102:
3919–3926
Schmidt M, Voutetakis A, Afione S et al (2008) Adeno-associated virus type 12 (AAV12): a novel AAV serotype with
sialic acid- and heparan sulfate proteoglycan-independent
transduction activity. J Virol 82:1399–1406
Smith RH, Yang L, Kotin RM et al (2008) Chromatographybased purification of adeno-associated virus. Methods Mol
Biol 434:37–54
Srivastava A (2008) Adeno-associated virus-mediated gene
transfer. J Cell Biochem May 23
Vandendriessche T, Thorrez L, Acosta-Sanchez A et al (2007)
Efficacy and safety of adeno-associated viral vectors based
on serotype 8 and 9 vs. lentiviral vectors for hemophilia B
gene therapy. J Thromb Haemost 5:16–24
Wang L, Herzog RW (2005) AAV-mediated gene transfer for
treatment of hemophilia. Curr Gene Ther 5:349–360
Xiao W, Chirmule N, Berta SC et al (1999) Gene therapy
vectors based on adeno-associated virus type 1. J Virol
73:3994–4003
Xie Q, Bu W, Bhatia S et al (2002) The atomic structure of
adeno-associated virus (AAV-2), a vector for human gene
therapy. Proc Natl Acad Sci 99:10405–10410
Ye C, Pintel DJ (2008) The transcription strategy of bovine
adeno-associated virus (B-AAV) combines features of
both adeno-associated virus type 2 (AAV2) and type 5
(AAV5). Virology 370:392–402
Zincarelli C, Soltys S, Rengo G et al (2008) Analysis of AAV
serotypes 1–9 mediated gene expression and tropism in
mice after systemic injection. Mol Ther 16:1073–1080