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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 864-874
REVIEW ARTICLE
Adeno-Associated Virus Vectors and Hematology
By
David W. Russell and
Mark A. Kay
From the Markey Molecular Medicine Center and the Department of
Medicine, University of Washington, Seattle, WA; and the Departments of
Pediatrics and Genetics, Stanford University, Stanford, CA.
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INTRODUCTION |
GENE THERAPISTS have a special fondness
for hematology. Initially, this was due to the many inherited diseases
that could potentially be cured by ex vivo genetic modification of hematopoietic stem cells. Once the early murine leukemia virus (MLV)
vectors were found to transduce only a small percentage of primate stem
cells, the search began for other types of vectors that might work more
efficiently, and adeno-associated virus (AAV) vectors were among the
first alternatives to be investigated. Although a substantial amount of
work with AAV vectors has occurred over the last several years, the
bulk of evidence suggests that they transduce hematopoietic stem cells
poorly, and that they will not replace retroviral vectors in stem cell
gene transfer applications. Instead, it is applications involving the
production of secreted proteins that now appear especially promising
for AAV vectors, where they are delivered by direct injection rather than ex vivo cell manipulation. Recent animal studies have shown that
AAV vectors encoding factor IX can lead to long-term, therapeutic clotting factor levels after in vivo administration, setting the stage
for what may well be a cure for hemophilia B in the near future. Animal
experiments have also shown that AAV vectors expressing erythropoietin
can produce dramatic increases in hematocrit levels. Here we will
review the role of AAV vectors in the field of hematology, including
the basic biology of AAV, vector transduction properties, their use in
hematopoietic cells, and their potential for delivering secreted
proteins such as clotting factors and cytokines.
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BIOLOGY OF AAV |
AAV is a dependent parvovirus with an  4.7-kb single-stranded linear
genome that contains two open reading frames (rep and cap).1 The genome encodes 4 replication (Rep)
proteins and 3 capsid proteins (VP1-3) (Fig
1). The inverted terminal repeats (ITRs) can pair to form
characteristic T-shaped hairpins, which are the only sequences required
in cis for replication and packaging. For a productive
infection to occur, AAV requires coinfection with a helper virus such
as adenovirus, which allows the viral genome to replicate episomally,
and leads to synthesis of the AAV proteins. In the absence of helper
virus, the AAV genome can integrate into the host cell chromosome,
where it remains in a latent proviral state until infection with helper
virus occurs, at which point the proviral genome excises and
replicates, and a productive infection resumes.
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| Fig 1.
Structure of wild-type and vector AAV genomes. (A) Map of
the wild-type AAV genome, including Rep (solid) and Cap (open) reading
frames, promoters (p5, p19, and p40), polyadenylation site (pA), and
inverted terminal repeats (ITR). The viral transcripts encoding the
different Rep and Cap (VP1-3) proteins are shown below the genome. The
smaller Rep proteins, VP2 and VP3, are translated from internal
initiation sites. (B) Map of a typical AAV vector, showing replacement
of the viral Rep and Cap genes with a transgene cassette (promoter,
transgene cDNA, and polyadenylation site). (C) Secondary structure of
the AAV ITR, with the locations of the Rep binding site (RBS) and
terminal resolution site (TRS) indicated.
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Several different serotypes of AAV have been identified by serological
analysis,2-5 and DNA sequencing has shown significant differences in their capsid genes that presumably account for the
distinct serological profiles.6-11 Antibodies against AAV types 1-3 and 5 are frequently found in human serum
samples.12-14 Virtually all the AAV vectors developed to
date were based on AAV type 2, which has implications for gene therapy,
because there is a high prevalence of neutralizing antibodies against
this serotype in human populations.14,15 The use of other
vector serotypes has the potential to overcome this immunity, and may
allow for repeat vector administration.8 Differences in
tissue tropism could also influence which cell types are susceptible to
transduction by each vector serotype.8-10 None of the AAV
serotypes have been shown to be pathogenic.
A fascinating feature of the AAV life cycle is the frequent integration
of proviral genomes at a common site on human chromosome 19 (19q13-qter).16-18 Although no significant homology exists
between this site-specific integration locus and AAV, the locus does
contain a binding site for the AAV Rep protein, which is required for the integration reaction.19-21 A number of research groups
have sought to capitalize on this feature by designing vectors that integrate at chromosome 19.22-26 It is not clear how useful
this approach will be, because the integration reaction is not
predictable, causes chromosomal rearrangements, and occurs at about the
same rate as random integration. Most AAV vectors do not include the rep gene (which would significantly limit the packaging
capacity), and they do not integrate at the chromosome 19 locus.27-32
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AAV TRANSDUCING VECTORS |
AAV vectors typically contain a transgene expression cassette flanked
by the viral ITRs (Fig 1). The conventional method for generating
vector stocks consists of cotransfection of a plasmid containing the
vector genome with a trans-acting helper construct for
rep and cap expression. The transfection is performed
in the presence of adenovirus, and 2 to 3 days later the cells are
lysed and the vector is harvested and purified. A major factor in AAV research has been the difficulty in preparing high-quality vector stocks, because the cotransfection method is cumbersome and
time-consuming. Recent improvements in vector production include
replacing adenovirus infection with a transfected construct containing
a select set of adenovirus genes,33 split rep and
cap expression cassettes to eliminate replication-competent AAV
(which frequently contaminates stocks),34 and the
development of higher titer packaging cell lines.35,36
Vector purification is critical, because residual helper virus could
significantly influence transgene expression levels,37,38
and the cellular debris contaminating crude preparations contains
transfected plasmids and transgene expression products that can mimic
true transduction events.39 Today's standards require that
AAV vector purification protocols include isopycnic centrifugation on
CsCl gradients (or perhaps column purification methods that are being
developed), and heat inactivation of remaining adenovirus.
An important consideration in analyzing vector stocks is the method
used for titering. Although functional titers (the amount of transgene
expression produced by a stock) are useful in predicting vector
performance, the number of vector particles required to produce a
single transgene expression event can vary from <100 to >10,000
depending on the vector, cell line, and/or production methods. To
obtain a reproducible titer measurement, one should determine vector
particle numbers directly by quantifying the number of vector genomes
present in purified vector preparations. We recommend performing
alkaline Southern blot analysis of DNA released from vector virions,
with quantitation of the signal from intact, full-length,
single-stranded vector genomes.36
Figure 2 outlines the steps involved in
transduction by AAV vectors. The vector virion interacts with cellular
receptor(s) to enter the cell. An early candidate for the AAV type 2 receptor was a 150-kD glycoprotein that bound to virus
particles40; however, subsequent studies have not
identified this protein. More recently, heparan sulfate proteoglycan
was shown to play a role in the binding and infection of AAV type
2,41 as was fibroblast growth factor receptor 1 (FGFR1)42 and  V 5 integrin.43 Heparan
sulfate is proposed to function as a primary receptor, with FGFR1 and V5 integrin acting as coreceptors. The exact roles and
interactions of these (and possibly other) molecules in virus
internalization have not been elucidated. Serotypes other than AAV type
2 may bind different molecules for entry, because they have
characteristic host ranges and competition experiments suggest they use
distinct receptors.8-10 After internalization, the particle
is rapidly transported to the nucleus and the vector genomes are
released. Little is known about these events, although it is clear that entering vector genomes and capsid proteins can be efficiently delivered to the nucleus, even in nondividing cells.27,44
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| Fig 2.
Transduction by AAV vectors. The different steps required
for transduction by AAV vectors are indicated, including an initial
interaction with a variety of possible receptor and coreceptor
molecules on the cell surface, virion internalization, nuclear entry
and release of the single-stranded vector genomes, followed by
second-strand DNA synthesis, hybridization of complementary input
genomes, and/or chromosomal integration before gene expression can
occur from a double-stranded template. Potential
secondary structures of episomal vector genomes are shown, as are
vector-encoded RNA molecules (bent arrows). Question marks indicate a
lack of detailed understanding of the particular step involved. For
further details see main text.
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Once in the nucleus, the entering single-stranded genomes are assumed
to be transcriptionally inactive, and additional events are required
before transgene expression occurs. Early experiments with selectable
markers showed that AAV vectors can stably integrate into host
chromosomes.45-48 Southern blot, polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH) analyses suggested
that vector integration occurred at random locations other than the
site-specific integration locus of chromosome
19.27,28,30,32 Cloning and sequencing of chromosomal
junction fragments from cells transduced with neo shuttle
vectors has shown that most vector genomes integrate by nonhomologous
recombination at random locations as single-copy proviruses with
terminal deletions.29,31 When preintegration sites were
analyzed, provirus insertion was found to be associated with
chromosomal translocations and/or deletions,31 raising
safety issues regarding the types of mutations that may be caused by
AAV vectors. It is not known if integration requires a double-stranded
or single-stranded vector molecule, nor if the vector deletions occur
before or during the recombination reaction (Fig 2). Because the vector
does not encode specific integration proteins, the reaction must use
host cell enzymes, and it requires a chromosomal break. Only a small
percentage of entering vector genomes ultimately
integrate,27 which is also true of wild-type viral
genomes,49-51 presumably due to limiting host cell factors.
It should be noted that these detailed integration studies were
performed on cultured cells undergoing selection in vitro, and the
results may not apply to all cell types. Several studies suggest
that vector proviruses can be found as large concatamers after
in vivo administration (see below).52-55
In addition to transduction by integration, transgene expression can
occur transiently from episomal AAV vector genomes.56,57 Double-stranded, episomal vector transcription templates could be
formed either by second-strand synthesis, or hybridization of
complementary input genomes, because either strand is packaged in the
virion (Fig 2). AAV vectors can also transduce cells by modifying
homologous chromosomal sequences in a gene-targeting process.58 Thus, the deceptively simple AAV vector genome
can transduce cells in several ways, and it is not always clear which mechanisms are involved in a particular experiment. Further
complicating factors are related to various host cell conditions that
can dramatically affect transduction rates, including S
phase,27 exposure to agents that induce DNA repair
functions,59,60 protein phosphorylation levels,61,62 expression of helper virus gene
products,37,38 and transgene silencing.63 Some
of these conditions presumably affect the cellular DNA synthesis or
repair processes important for transduction, including second-strand
synthesis and/or provirus integration. The ultimate fate of entering
vector genomes will depend on the type of cell being transduced, its
metabolic and proliferative state, and the transduction conditions used.
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TRANSDUCTION BY AAV VECTORS IN VIVO |
Long-term transgene expression has been produced by AAV vectors in
several tissues after in vivo administration.52-54,64-75
Attempts to determine the structure of the AAV vector genome in target tissues transduced in vivo have typically shown the presence of high-molecular-weight concatamers.52-55,74,76 Conversion of
input single-stranded vector genomes to high-molecular-weight
double-stranded concatamers is associated with an increase in transgene
expression,55,76 which can take several weeks after in vivo
administration until steady-state levels are
reached.52,70,72,73,76,77 A similar, slow increase in
transgene expression can also occur in vitro, especially with quiescent
cells.27,45
Long-term transgene expression could be produced by integrated
concatamers, or by stable concatameric episomes (especially in
nondividing cell populations), and there is evidence for both scenarios.55,76,78 Recently, Miao et al55
studied the kinetic fate of AAV vector genomes during the process of
liver transduction in mice. The single-stranded DNA genomes disappeared
over a 5-week period with a concomitant increase in double-stranded,
high-molecular-weight forms. Pulse-field gel and FISH analyses were
used to establish that the vector concatamers were integrated in about
5% of cells. This correlates well with the number of cells shown to
express vector-encoded RNA or protein.70 In muscle
experiments, Duan et al76 observed circular, monomeric
vector episomes that were converted to larger multimers over an 80-day
period, and the stability of these episomes was enhanced by the AAV
ITRs. It is not known if the multimeric circles ultimately integrate.
Further studies are needed to clarify the relationship between episomal
and integrated concatameric forms, and whether tissue-specific factors
influence transduction pathways.
Vector genome concatamers could be formed by replication or ligation
and/or recombination of input monomers (which are often present at high
multiplicity). In the many cases where stationary phase cells have been
transduced in vivo, vector genome replication would have been limited
to those cells undergoing unscheduled DNA synthesis by host enzymes,
while vector genome ligation and recombination may have been promoted
by the high multiplicities of infection (MOIs) used for in
vivo experiments. The concatamer formation consistently observed in
vivo stands in contrast to the formation of single-copy, integrated
proviruses often observed in vitro.29,31 Potential factors
that could favor concatamer formation include the use of normal,
nonproliferating cells that do not dilute input vector genomes, a long
time course in the absence of selection, and/or the presence of
replication-competent AAV particles in vector stocks.
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TRANSDUCTION OF HEMATOPOIETIC CELLS BY AAV VECTORS |
Several research groups have reported transduction of various types of
hematopoietic cells by AAV vectors. The evidence is clear that
transformed, hematopoietic cell lines can be
transduced,30,46,79-81 although the transduction rates are
significantly lower than those of many nonhematopoietic cell types
infected at the same MOI.8,57,82 In the case of primary
hematopoietic cells, the efficacy of AAV vectors is controversial, as
illustrated by studies with hematopoietic progenitors. Several
investigators have concluded that progenitors can be efficiently
transduced by AAV vectors.83-92 It is difficult to
reconcile some of these studies with recent reports showing that
transduction of CD34+ progenitors requires extremely high
MOIs of 106 to 108 vector particles/cell, and
often leads to transient transgene expression.57,82 Still
other investigators have been unable to transduce hematopoietic
progenitors, and these experiments have not been published. For
example, we could not detect transduction of hematopoietic progenitors
by AAV vectors at MOIs up to 106 vector particles/cell
(D.W.R. and R.K. Hirata, unpublished results).
Despite a decade of research in this field, only 2 transplantation
studies have been published to determine if hematopoietic stem cells
can be transduced by AAV vectors. Ponnazhagan et al91 described murine transplantation experiments with AAV vectors and
reported long-term persistence of vector DNA in bone marrow and spleen
specimens as detected by PCR (including samples from 1 secondary
transplant recipient). Although this is the strongest evidence to date
for murine stem cell transduction, the cell types containing vector
sequences were not identified and progenitor assays were not presented
to document the persistence of transduced hematopoietic cells, making
it difficult to accurately assess stem cell transduction rates.
Furthermore, the MOIs of 1 and 10 used were 5 to 7 logs below what
others have shown are required for progenitor
transduction.57,82
Schimmenti et al93 recently described a transplantation
study with rhesus monkeys, in which hematopoietic cells (including granulocytes and lymphocytes) containing vector sequences could be
detected for over 15 months after reinfusion of CD34+ cells
transduced at MOIs >1,000 physical AAV vector particles/cell. No
transgene expression data were presented. The transduction rates were
low (approximately 1 in 105 cells), suggesting that AAV
vectors are less effective than retroviral vectors for the transduction
of primate hematopoietic stem cells. Further studies will be required
to resolve remaining questions, such as whether murine stem cells are
transduced at higher rates than primate stem cells by AAV vectors (as
appears true for retroviral vectors), and whether higher MOIs would
improve stem cell transduction rates.
There are several possible explanations for the conflicting results
obtained with primary hematopoietic cells, including differences in
vector design, stock preparation, the cell types being analyzed, transduction conditions, and transgene assays. One possibility is that
the stocks used in many of the experiments reporting high transduction
rates were crude cell lysates, which contain cellular debris,
adenovirus particles, transgene DNA, RNA, and protein molecules, in
addition to recombinant AAV. These contaminating substances could have
influenced transduction rates, or even produced false-positive results
themselves in the assays used to monitor transgene
expression.39 Another important factor is the cell population being studied. We have found that the bone marrow stromal cells present in hematopoietic cell preparations are transduced at high
rates by AAV vectors (D.W.R. and R.K. Hirata, unpublished results), so one must be careful to exclude these (and
other) contaminating cells from the analysis when assays other than
progenitor colony formation are used. Variation in the transduction
rates of CD34+ cells from different individuals must also
be considered.94 The particular transgene assay being used
can have significant effects on the apparent "transduction rate."
Assays that detect transient transgene expression are likely to produce
higher transduction rates than those requiring stable
integration.56,57,82 Assays that detect vector DNA
sequences such as PCR or Southern analysis are much more sensitive than
those requiring transgene expression, because most entering vector
genomes remain as transcriptionally inactive, single-stranded
molecules.27 At the very high MOIs used in some
experiments, the surrounding culture media itself is likely to contain
many vector particles. A careful consideration of these experimental
factors can help in comparing the different published studies.
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GENE THERAPY FOR HEMOPHILIA |
One of the most promising applications of AAV vectors is the treatment
of hemophilia. This is due in part to the well-understood genetics of
the disease, easily measured clinical endpoints, and the availability
of mouse95-97 and canine98-100 animal models.
Patients with hemophilia are categorized by degree of severity, with
less than 1% of normal clotting factor levels producing severe, 1% to
5% moderate, and 5% to 20% mild disease. Thus, reconstitution of as
little as 1% of clotting factor can convert a severe phenotype to a
moderate one, and there is a broad range of potentially therapeutic factor levels, which significantly improves prospects for successful gene therapy. For a recent review of hemophilia gene therapy see Herzog
and High.101
Even though hemophilia A and B are indistinguishable clinically, their
gene therapy strategies are not necessarily the same. The relative
amount of factor VIII that needs to be synthesized is much less than
factor IX. Based on the molecular weight, normal plasma concentrations,
and differences in the volume of distribution, we estimate that this
difference is at least 2 orders of magnitude. The length of the factor
IX coding region is about 1.4 kb, which is well below the 4.5 kb
packaging limit of AAV vectors, while the full-length factor VIII cDNA
of about 7 kb exceeds the packaging limit. Even the B-domain-deleted
factor VIII message is about 4.4 kb, making it very difficult to
produce a high-titer AAV vector with a promoter and polyadenylation
site. An alternative strategy is to split the factor VIII molecule into
2 chains, and use separate AAV vectors. This approach will require
efficient delivery of both vectors in the proper ratio to target cells,
as well as functional reassembly of the two factor VIII peptides.
Although AAV vectors can transduce many tissues in vivo, the efforts
for hemophilia to date include muscle and liver experiments, the latter
being a natural site of factor VIII and IX production. Other tissues such as vascular endothelium may also be appropriate targets. Although
factor IX can be produced in muscle cells (see below), it is not
completely clear what the biological consequences are of ectopic
clotting factor synthesis, especially as it relates to the functional
activity of the protein and possible inhibitor formation. It appears
less likely that factor VIII can be efficiently produced in
muscle.102,103
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PRECLINICAL STUDIES WITH AAV VECTORS FOR HEMOPHILIA B |
Significant progress has been made with AAV vectors encoding factor IX.
Koeberl et al68 were the first to show that AAV vectors
could be used for the expression of human factor IX in vivo, by
delivering the vector to mouse liver after gamma irradiation. Irradiation was used in these experiments to induce DNA repair functions that were found to increase transduction rates in
vitro.59 The amount of factor IX produced was less than 1 ng/mL, or about 0.01% of the normal level of 5 µg/mL. Two groups
shortly thereafter achieved therapeutic human factor IX levels after in
vivo delivery of AAV vectors in mice. Herzog et al72
delivered an intramuscular injection of 2 × 1011
physical particles of an AAV vector containing the cytomegalovirus
(CMV) enhancer-promoter driving the human factor IX cDNA and produced
plasma factor IX levels as high as 300 ng/mL that lasted for the length
of the study. Snyder et al70 administered 8.4 × 1010 particles of an AAV vector containing the human
factor IX cDNA driven by a Moloney leukemia virus (MLV) long terminal
repeat (LTR) promoter into the livers of mice via portal vein infusion,
and produced up to 2,000 ng/mL of biologically active factor IX (or
40% of normal levels), an amount considered curative. With portal vein
infusion, about 5% of hepatocytes were positive for AAV-mediated gene
expression as determined by RNA in situ hybridization and
immunohistochemical staining for a marker gene. By quantitative
Southern blot analysis there was on average more than 1 AAV vector
genome copy per cell. High-level factor IX gene expression of up to
1,000 ng/mL was also achieved by Nakai et al,104 who
transduced mouse livers with 1 × 1011 particles of an
AAV vector containing the elongation factor 1 promoter via portal
vein injection. Interestingly, no factor IX expression was observed
when the CMV promoter was used in the liver70,104, as also
observed with retroviral vectors,105 emphasizing the importance of promoter choice as related to target organ. In the more
recent studies, high-level expression in liver and muscle was achieved
without pretreatment of the target organ to increase transduction
rates, perhaps because of the higher MOIs or different expression
cassettes used.
Three recent studies have shown the therapeutic value of AAV-mediated
gene transfer in murine and/or canine hemophilia B106-108 (earlier studies had used normal mice transduced with the human gene).
These animals have severe hemophilia with virtually no factor IX
activity. In the first study, 2.5 × 1012 AAV vector particles expressing human factor IX from the CMV promoter were administered by intramuscular injection to a dog with hemophilia B.106 A 1-week partial reduction in the whole-blood
clotting time was observed. The transient nature of the clinical
improvement was likely caused by the inhibitor formed against the human
factor IX protein. A second study showed up to 70 ng/mL of plasma
factor IX after intramuscular delivery of 3 to 8 × 1012 particles/kg of an AAV vector expressing canine factor
IX from the CMV promoter to hemophilia B dogs.107 This
plasma level represents about 1% of normal and would be of therapeutic
benefit to a severe hemophiliac. One of 5 animals had a transient
inhibitor that resolved without sequelae. These animals had decreases
in their whole-blood clotting and partial thromboplastin times.
In a liver study, AAV vectors expressing human factor IX from an MLV
LTR promoter were infused into the tails or portal veins of hemophilia
B mice.108 With a dose of 6 × 1010
particles, the bleeding time was reduced to the normal range of 3 to 5 minutes from a pretreatment level of over 30 minutes and was associated with a plasma factor IX concentration of 250 to 1,800 ng/mL. This correlated with a human factor IX bioactivity of up to 100%. In the
same study, a similar AAV vector encoding canine factor IX was given to
hemophilia B dogs at about 1/10 the dose given to the mice (based on
body weight; about 2 × 1011 particles/kg), which
produced persistent expression of canine factor IX and reduced whole-blood clotting and partial thromboplastin times. One animal had
1% of the normal factor IX plasma level. The untreated animals have on
average 5 spontaneous bleeding episodes per year, and at the time of
publication, only 1 treated animal had experienced a spontaneous bleed,
where 6 or 7 in total would have been expected. There were no
inhibitors associated with the therapy.
There are a number of issues relating to muscle versus liver as target
organs for AAV factor IX vectors. Although the muscle is more readily
accessible than the liver, in humans the portal vasculature can be
infused by nonsurgical, radiographic procedures, making either organ a
reasonable approach for clinical trials. Most adult hemophiliacs have
been infected with hepatitis viruses, so the complications of liver
disease may influence hepatic therapy outcomes. However, newer factor
replacement therapies have substantially decreased the incidence of
hepatitis in the pediatric hemophilia population, making this less of
an issue in the future. The potency of AAV-mediated gene expression
appears to be in favor of the liver. The relative AAV vector dose
required to achieve similar factor IX levels was 40 times greater in
the muscle than the liver. This point is complicated by the fact that
the vectors used were produced in different labs, and contained
different expression cassettes. Moreover, it is important to realize
that promoter optimization may allow for increased expression in liver
and/or muscle. Other factors that could differ between these organs are their capacity for posttranslational protein modifications essential for clotting factor activity, antigen presentation properties, and
natural rates of cellular turnover and replacement.
The clinical endpoints required to monitor gene therapy for hemophilia
are straightforward, because the severity of the disease is directly
related to the factor activity measured in the patient's blood. A
major remaining issue is related to inhibitor formation. To minimize
this risk, the first patients to be treated should have a long history
of parenteral protein replacement without inhibitor formation. However,
this does not guarantee the absence of inhibitors, because antigen
presentation from soluble proteins may be different than from proteins
produced and secreted by cells, ectopic clotting factor production
could generate a novel immune response, the pharmacokinetics of
continuous factor expression versus periodic infusion could affect
antibody formation, and there may also be immunologic consequences of
the vector particle. It is possible that a patient receiving an AAV
factor IX vector could develop a new inhibitor, and become more
difficult to manage by clotting factor transfusions in the future,
which needs to be considered in designing clinical trials. Although the
data in hemophilia B dogs suggest that AAV vectors can express factor IX without leading to inhibitor formation, many of these questions can
only be resolved by appropriate gene therapy trials in people. After
the initial patients are treated, the treatment of patients prone to
inhibitor formation will need to be addressed. Given the convincing
preclinical data showing that long-term therapeutic factor IX levels
can be achieved, successful gene therapy for hemophilia B with AAV
vectors may soon become reality.
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COMPARING VECTORS FOR THE TREATMENT OF HEMOPHILIA |
Many different vector systems have been used in various tissues in
preclinical studies for the treatment of hemophilia, but for a
comparison to AAV vectors we will limit our discussion to in vivo
studies of the muscle and liver. Retroviral and adenoviral vectors
encoding factors VIII and IX have been used for gene transfer into
muscle and liver.95,109-111 When recombinant retroviruses
were infused into the portal vein of hemophilia B dogs,105
partial hepatectomy was required to stimulate hepatocellular
proliferation, a prerequisite for MLV retroviral transduction.
Expression persisted for at least a year, enough to lower the whole
blood clotting time by about 50%. However, only about 1% of
hepatocytes contained proviral DNA and factor IX expression was in the
range of 0.1% of normal and not considered therapeutic. Moreover,
surgical partial hepatectomy would not be appropriate for clinical
trials in humans. More recently, improved rates of retroviral gene
transfer were achieved in mice using growth factors to induce liver
regeneration in place of liver injury.112,113 Under some
conditions, as many as 30% of the hepatocytes were transduced. In
addition, there is some evidence to suggest lentiviral vectors will
transduce nondividing cells, including hepatocytes.114
These methods have yet to be assessed in preclinical studies of
hemophilia, and one must be careful in extrapolating in vivo results
with retroviral vectors tested in animals to humans, because many
retroviral vectors are inactivated by human serum.115,116
First-generation recombinant adenoviruses have been used in mice to
achieve therapeutic levels of factors VIII and
IX.95,109,111,117-119 In some strains, persistent
expression has been achieved with long-term correction of murine
hemophilia. However, the same adenoviral canine factor IX vector that
was shown to persist in mice produced only transient, supra-normal
factor levels with concomitant correction of the bleeding diathesis in
hemophilia B dogs, due to the toxicity and immunogenicity of the
vector.111 The discrepancy between the mouse and dog is
related to differences in the immune responses seen in different inbred
strains of mice.120 Transient expression of human factor
VIII in canine hemophilia A has been obtained using a recombinant
adenovirus; however, the factor VIII-deficient dogs made inhibitors to
the human protein.118 Newer generation, "gutted"
adenoviral vectors containing fewer viral genes show decreased toxicity
and immunogenicity, but their persistence has not yet been shown in
preclinical studies of genetic diseases such as
hemophilia.121,122
In the case of hemophilia B, AAV vectors show the greatest potential at
present, with several studies documenting long-term, therapeutic factor
IX levels in animals. The lack of significant inflammatory response
caused by the vector, the potential for permanent genetic modification
by chromosomal integration, and the ability to transduce quiescent
cells are major advantages of AAV vectors. Although retroviral vectors
integrate, only the newer vectors based on alternative retroviruses
such as lentiviruses have the potential to transduce nonproliferating
cells in liver or muscle tissue. Adenovirus vectors efficiently
transduce these cell types; however, they do not integrate and can be
associated with a significant immune response. The newer,
"gutted" adenovirus vectors may prove more effective, and at
least in quiescent cells, it is possible that an episomal vector genome
could persist long-term. A major disadvantage of AAV vectors is their
limited packaging capacity, which may prevent their use in delivering
factor VIII or other genes with cDNAs over 4 kb. As research on gene
therapy for hemophilia progresses, the advantages and disadvantages of these and newer vector systems will become apparent, and the choice of
vector will ultimately depend on the particular clinical application.
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GENE THERAPY FOR OTHER COAGULOPATHIES |
There are other coagulation disorders being studied in preclinical gene
therapy experiments. Factor X deficiency is a rare but serious bleeding
diathesis. Retroviral-mediated hepatic gene transfer into rats has
resulted in persistent levels of factor X that are 10% to 43% of
normal.123 Although AAV vectors have not been tested for
factor X deficiency, the coding sequence is similar in size to factor
IX, making this an excellent candidate for AAV-mediated gene transfer.
Gene therapy could also be used to treat hypercoagulation disorders,
such as protein C or S deficiency. Unlike the bleeding disorders,
individuals hemizygous for either gene are symptomatic, with recurrent
thromboses. It is believed that over 5% of individuals with recurrent
thromboembolic events have inherited protein C or S deficiency. Because
these individuals have 1 normal allele, clinical manifestations can
occur with 50% of the normal protein level. Thus, very high transgene
expression levels will be required to produce a clinical effect in
heterozygous individuals. Rare, homozygous patients (about 1/500,000
births) can have neonatal purpura fulminans or disseminated
intravascular coagulation, and might have marked clinical improvement
with smaller amounts of gene reconstitution. Cai et al124
have shown retroviral-mediated gene transfer of protein C into rat
hepatocytes in vivo with up to 22% of the normal plasma concentration.
The high level of protein produced may in part have been caused by the
formation of nonneutralizing antibodies against the human protein that
prolonged the half-life of protein C. Both the protein C and S cDNAs
will fit in AAV vectors, so these diseases could also be candidates for
AAV-mediated gene therapy. However, in addition to the very high
transgene expression levels required, a therapeutic response could be
difficult to assess in these patients, because it may require prolonged
observation before a decrease in thomboses can be documented. This will
complicate early clinical trials, and could delay the development of
genetic therapies for these disorders.
| |
AAV AND OTHER HEMATOLOGIC DISEASES |
The success of preclinical studies for hemophilia B with AAV vectors
suggests that they could be used for the production of a variety of
secreted proteins. Although these include several cytokines that might
be used to influence hematopoiesis, the best studied example is the use
of AAV vectors for the production of erythropoietin to treat anemia.
This protein is normally made in the kidney with small amounts being
produced in the liver, and patients with chronic renal disease or
possibly thalassemia might benefit from erythropoietin gene therapy.
Several groups have shown sustained expression of erythropoietin in
primates and/or rodents after intramuscular injection of AAV
vectors.35,67,125-128 In these studies, persistent
hematocrit elevations were produced and, in some cases, the levels were
dangerously high and phlebotomy was required. Thus, the success and
safety of erythropoietin therapy will require regulated transgene
expression. Three studies have shown that such regulation is possible
by codelivery of inducible transcription factors that act on the
vector-encoded erythropoietin transgene. Systems based on
tetracycline-regulated transactivation127,128 and
rapamycin-regulated chimeric transcription factors126 have been used.
Although erythropoietin expression can clearly be regulated with
appropriate doses of tetracycline or rapamycin, delivery of
transcription factor genes raises additional safety concerns. It is
difficult to predict what effects an exogenous transcription factor
will have on the regulation of endogenous genes, either through direct
binding at chromosomal loci, or through interactions with cellular
proteins. Recombination of the vector-encoded transcription factor gene
with cellular genes could also generate new fusion proteins that might
influence gene expression. In addition, the regulation is not
physiologic, so the maintenance of therapeutic transgene levels
requires continuous clinical monitoring with frequent dose adjustments
of the appropriate regulatory drug. These issues are especially
relevant for erythropoietin gene therapy, because too much expression
could produce fatal polycythemia, while safe and effective
pharmacological administration of the erythropoietin protein is already
possible. Some of these problems might be solved by using an endogenous
promoter that responds in a physiologically relevant manner to
anemia129,130; however, proper regulation could still be
difficult because of chromosomal position effects. Appropriate
preclinical safety and efficacy studies will need to be completed
before these strategies are applied clinically.
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CONCLUSIONS |
The last few years have seen remarkable advances in the development of
AAV vectors for gene therapy, especially for applications in
hematology. Although early enthusiasm for their use in hematopoietic cells has not been followed by reports of potentially therapeutic stem
cell transduction rates, their use in liver or muscle can clearly
generate physiologically relevant levels of secreted clotting factors
or cytokines. Several studies have shown that therapeutic factor IX
levels can be produced by AAV vectors in animals, and there is every
reason to believe the same will be true of humans. The next few years
should see the first clinical trials of AAV vectors for hemophilia B
and, hopefully, a permanent genetic cure for these patients.
| |
FOOTNOTES |
Submitted February 10, 1999; accepted April 21, 1999.
Supported by grants from the March of Dimes Birth Defects Foundation
and the US National Institutes of Health (Nos. HL53750 and HL53682).
Address reprint requests to David W. Russell, MD, PhD, Division
of Hematology, Department of Medicine, Mailstop 357720, Room
K-236A HSB, University of Washington, Seattle, WA 98195-7720; e-mail:
drussell{at}u.washington.edu.
| |
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INTRODUCTION
BIOLOGY OF AAV