Current Gene Therapy, Volume 5, No. 3, 2005
Contents
Recombinant Adeno-Associated
Virus: Current Achievements and Limitations
Guest
Editor: Philippe Moullier
Editorial Pp.263-263
Helper Functions Required for Wild Type and
Recombinant Adeno-Associated Virus Growth Pp.265-271
Marie-Claude
Geoffroy and Anna Salvetti
Mechanisms of Adeno-Associated Virus Genome
Encapsidation Pp.273-284
Jennifer
Timpe, Joyce Bevington, John Casper, John D. Dignam and James P. Trempe
New Recombinant Serotypes of AAV Vectors Pp.285-297
Guangping
Gao, Luk H. Vandenberghe and James M. Wilson
AAV Hybrid Serotypes: Improved Vectors for
Gene Delivery Pp.299-310
Vivian
W. Choi, Douglas M. McCarty and R. Jude Samulski
Adeno-Associated Viral Vectors for Clinical
Gene Transfer Studies Pp.311-321
Richard
O. Snyder and Joyce Francis
Immune Responses to Adeno-Associated Virus
Vectors Pp.323-331
Anne
K. Zaiss and Daniel A. Muruve
Adeno-Associated Virus (AAV) Vectors in the
CNS Pp.333-338
Thomas
J. McCown
Adeno-Associated Viral Vectors for Retinal
Gene Transfer and Treatment of Retinal Diseases Pp.339-348
Alberto
Auricchio and Fabienne Rolling
AAV-Mediated Gene Transfer for Treatment of
Hemophilia Pp.349-360
Lixin
Wang and Roland W. Herzog
Recent Developments in Recombinant
AAV-Mediated Gene Therapy for Lung Diseases Pp.361-366
Terence
R. Flotte
Abstracts
[Back to top] Editorial
Philippe
Moullier
This special issue
was initially meant to help graduate students entering the field of gene
therapy by providing a comprehensive review of recombinant adeno-associated
virus (rAAV) from virology to current clinical applications. However, each article
is of such a high quality that I believe this issue will also provide a useful
document for the entire gene therapy community.
Although wild type
AAV was studied for decades, Xiao Xiao and Jude R. Samulski’s paper in 1996
represents the first evidence that rAAV produced efficient and long-term gene
transfer in a mouse after a single in situ administration (i.e. the
skeletal muscle) [Xiao et al., 1996]. 1996 was also the year of the
lentivirus vector [Naldini et al., 1996]. Yet, 1996 was only one year after
the Orkin and Motuslky report [Orkin et al., 1995] emphasizing the need
for better vectors!
Today progress in
rAAV-mediated gene transfer is so spectacular that long-term, efficient, and
regulatable transgene expression is reproducibly achieved in large animal
models. For example, i) the entire limb of hemophilia dogs can be efficiently
transduced resulting in long-term phenotypic correction [Arruda et al.,
2004]; ii) rAAV administered once in nonhuman primate muscle shows sustained
regulatable transgene expression for more than 6 years [Rivera et al., 2005].
Simultaneously, the discovery of new AAV serotypes along with the ability to
encapsidate either “self-complementing” or “single-stranded” vector DNA
[McCarty et al., 2001] has turned this vector system into an extremely
powerful and versatile tool with preferential organ transduction patterns
depending on the AAV capsid origin and/or the vector DNA used. Finally,
considerable improvements have been made in the availability of clinical grade
rAAV stocks [Snyder et al., 2002], a critical issue, even though large-scale
production remains problematic.
On behalf of the Current
Gene Therapy journal, I would like to thank the authors for their
outstanding articles that truly illustrate the impressive number of
rAAV-related breakthroughs achieved in the past decade, although with no clear
evidence yet that this may be an effective therapeutic approach in humans.
[Back to top] Helper Functions Required for Wild Type and Recombinant
Adeno-Associated Virus Growth
Marie-Claude
Geoffroy and Anna Salvetti
The human
parvovirus Adeno-Associated virus (AAV-2) has been classified as a Dependovirus
because it requires the presence of a helper virus to achieve a productive replication
cycle. Several viruses such as Adenovirus (Ad), Herpes Simplex Virus (HSV),
Vaccinia virus, and human papillomaviruses (HPV) can provide the helper
activities required for AAV growth. The studies on the helper activities
provided by adenovirus have provided useful information not only to understand
the AAV-2 biology but also to develop tools for the production of recombinant
AAV particles (rAAV). This review will focus on the current knowledge about the
helper activities provided by the most extensively studied helper viruses, Ad
and HSV-1, and also illustrate the methods used to supply the helper functions
rAAV assembly.
[Back to top] Mechanisms of Adeno-Associated Virus Genome
Encapsidation
Jennifer
Timpe, Joyce Bevington, John Casper, John D. Dignam and James P. Trempe
The defective
parvovirus, adeno-associated virus (AAV), is under close scrutiny as a human
gene therapy vector. AAV’s non-pathogenic character, reliance on helper virus
co-infection for replication and wide tissue tropism, make it an appealing
vector system. The virus’ simplicity and ability to generate high titer vector
preparations have contributed to its wide spread use in the gene therapy
community. The single stranded AAV DNA genome is encased in a 20-25 nm
diameter, icosahedral protein capsid. Assembly of AAV occurs in two distinct
phases. First, the three capsid proteins, VP1- 3, are rapidly synthesized and
assembled into an empty virion in the nucleus. In the second, rate-limiting
phase, singlestrand genomic DNA is inserted into pre-formed capsids. Our
rudimentary knowledge of these two phases comes from radioactive labeling
pulse-chase experiments, cellular fractionation and immunocytological analysis
of infected cells. Although the overall pattern of virus assembly and
encapsidation is known, the biochemical mechanisms involved in these processes
are not understood. Elucidation of the processes of capsid assembly and
encapsidation may lead to improved vector production. While all of the parvoviruses
share the characteristic icosahedral particle, differences in their surface
topologies dictate different receptor binding and tissue tropism. Based on the
analysis of the molecular structures of the parvoviruses and capsid mutagenesis
studies, investigators have manipulated the capsid to change tissue tropism and
to target different cell types, thus expanding the targeting potential of AAV
vectors.
[Back to top] New Recombinant Serotypes of AAV Vectors
Guangping
Gao, Luk H. Vandenberghe and James M. Wilson
AAV based vectors
can achieve stable gene transfer with minimal vector related toxicities. AAV
serotype 2 (AAV2) is the first AAV that was vectored for gene transfer
applications. However, the restricted tissue tropism of AAV and its low
transduction efficiency have limited its further development as vector. Recent
studies using vectors derived from alternative AAV serotypes such as AAV1, 4, 5
and 6 have shown improved potency and broadened tropism of the AAV vector by
packaging the same vector genome with different AAV capsids. In an attempt to
search for potent AAV vectors with enhanced performance profiles, molecular
techniques were employed for the detection and isolation of endogenous AAVs
from a variety of human and non-human primate (NHP) tissues. A family of novel
primate AAVs consisting of 110 non-redundant species of proviral sequences was
discovered and turned to be prevalent in 18-19% of the tissues evaluated.
Phylogenetic and functional analyses revealed that primate AAVs are segregated
into clades based on phylogenetic relatedness. The members within a clade share
functional and serological properties. Initial evaluation in mouse models of
vectors based on these novel AAVs for tissue tropism and gene transfer potency
led to the identification of some vector with improved gene transfer to
different target tissues. Gene therapy treatment of several mouse and canine
models with novel AAV vectors achieved long term phenotypic corrections.
Vectors based on new primate AAVs could become the next generation of efficient
gene transfer vehicles for various gene therapy applications.
[Back to
top] AAV Hybrid Serotypes: Improved Vectors for
Gene Delivery
Vivian W. Choi, Douglas
M. McCarty and R. Jude Samulski
In recent years,
significant efforts have been made on studying and engineering adeno-associated
virus (AAV) capsid, in order to increase efficiency in targeting specific cell
types that are non-permissive to wild type (wt) viruses and to improve efficacy
in infecting only the cell type of interest. With our previous knowledge of the
viral properties of the naturally occurring serotypes and the elucidation of
their capsid structures, we can now generate capsid mutants, or hybrid
serotypes, by various methods and strategies. In this review, we summarize the
studies performed on AAV retargeting, and categorize the available hybrid
serotypes to date, based on the type of modification: 1) transcapsidation, 2)
adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4)
chimeric capsid. 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.
[Back to top] Adeno-Associated Viral Vectors for Clinical
Gene Transfer Studies
Richard
O. Snyder and Joyce Francis
Recombinant
adeno-associated viral (rAAV) vectors can mediate the safe and long-term
correction of genetic diseases in animal models following a single
administration. These pre-clinical studies are the basis of human trials that
have shown rAAV vector persistence and safety in humans following delivery to
lung, sinus, skeletal muscle, brain and liver. Transient disease correction has
also been demonstrated in humans treated for hemophilia B and cystic fibrosis
using AAV2 vectors. The physiochemical properties of rAAV vector virions are
amenable to industry accepted manufacturing methodologies, long-term storage
and direct in vivo administration. Recombinant adeno-associated virus
vectors are manufactured in compliance with current Good Manufacturing
Practices (cGMPs) as outlined in the Code of Federal Regulations (21CFR). To
meet these requirements, manufacturing controls and quality systems are
established, including 1) adequate facilities and equipment, 2) personnel who
have relevant education or experience and are trained for specific assigned
duties, 3) raw materials that are qualified for use and 4) a process (including
production, purification, formulation, filling, storage and shipping) that is
controlled, aseptic, reliable and consistent. Quality systems including Quality
Control (QC) and Quality Assurance (QA) are also implemented. These
manufacturing procedures and quality systems are designed so the product meets
its release specifications to ensure that patients receive a safe, pure, potent
and stable investigational drug.
[Back to top] Immune Responses to Adeno-Associated Virus
Vectors
Anne
K. Zaiss and Daniel A. Muruve
One of the biggest
challenges in optimizing viral vectors for gene therapy relates to the immune
response of the host. Adeno-associated virus (AAV) vectors are associated with
low immunogenicity and toxicity, resulting in vector persistence and long-term
transgene expression. The inability of AAV vectors to efficiently transduce or
activate antigen presenting cells (APCs) may account for their decreased
immunogenicity. AAV mediated gene therapy however, leads to the development of
antibodies against the vector capsid. Anti-AAV antibodies have neutralizing
effects that decrease the efficiency of in vivo gene therapy and can
prevent vector re-administration. Furthermore, recent studies have shown that
AAV vectors can elicit both cellular and humoral immune responses against the transgene
product. Both cell-mediated response and humoral response to the delivered gene
depend on a number of variables; including the nature of the transgene, the
promoter used, the route and site of administration, vector dose and host
factors. The response of the host to the vector, in terms of antigen-specific
immunity, will play a substantial role in clinical outcome. It is therefore
important to understand both, why AAV vectors are able to escape immunity and
the circumstances and mechanisms that lead to the induction of immune
responses. This review will summarize innate and adaptive immune responses to
AAV vectors, discuss possible mechanisms and outline strategies, such as capsid
modifications, use of alternative serotypes, or immunosuppression, which have
been used to circumvent them.
[Back to top] Adeno-Associated Virus (AAV) Vectors in the
CNS
Thomas J. McCown
Adeno-associated
virus (AAV) vectors exhibit a number of properties that have made this vector
system an excellent choice for both CNS gene therapy and basic neurobiological
investigations. In vivo, the preponderance of AAV vector transduction
occurs in neurons where it is possible to obtain long-term, stable gene
expression with very little accompanying toxicity. Promoter selection, however,
significantly influences the pattern and longevity of neuronal transduction
distinct from the tropism inherent to AAV vectors. AAV vectors have
successfully manipulated CNS function using a wide variety of approaches including
expression of foreign genes, expression of endogenous genes, expression of
antisense RNA and expression of RNAi. With the discovery and characterization
of different AAV serotypes, the potential patterns of in vivo vector
transduction have been expanded substantially, offering alternatives to the
more studied AAV 2 serotype. Furthermore, the development of specific AAV
chimeras offers the potential to further refine targeting strategies. These
different AAV serotypes also provide a solution to the immune silencing that
proves to be a realistic likelihood given broad exposure of the human
population to the AAV 2 serotype. These advantageous CNS properties of AAV
vectors have fostered a wide range of clinically relevant applications
including Parkinson’s disease, lysosomal storage diseases, Canavan’s disease,
epilepsy, Huntington’s disease and ALS. Each individual application, however,
presents a unique set of challenges that must be solved in order to attain
clinically effective gene therapies.
[Back to top] Adeno-Associated Viral Vectors for Retinal
Gene Transfer and Treatment of Retinal Diseases
Alberto
Auricchio and Fabienne Rolling
Retinal gene
transfer holds big promises for the treatment of inherited and non-inherited
blinding diseases, such as retinitis pigmentosa or age-related macular
degeneration. Key to the development of successful gene-based therapies for the
eye are efficient tools for retinal gene transfer. Vectors based on
adeno-associated viruses (AAV) are able to transduce robustly and persistently
different retinal cell types of animal models after a single intraocular
administration. Recombinant AAV (rAAV) vectors are versatile gene transfer
tools in that capsid proteins from dozens of AAV serotypes can be easily
interchanged, resulting in the creation of recombinant vectors with unique
transduction properties. This has allowed successful proof-of-principle studies
using rAAV-mediated gene transfer to restore retinal morphology and function in
small and large animal models of retinal diseases. In addition, gene delivery
using rAAV vectors in the eye seems to have appropriate biosafety
characteristics to rapidly move it from bench to bedside. All the above aspects
will be reviewed and discussed in detail below.
[Back to top] AAV-Mediated Gene Transfer for Treatment of
Hemophilia
Lixin
Wang and Roland W. Herzog
Adeno-associated
viral (AAV) gene transfer of coagulation factor VIII and IX to skeletal muscle
and liver of murine and canine models of hemophilia A and B have resulted in
sustained systemic expression and, in several studies, in complete cure of the
bleeding disorder. These impressive results prompted initiation of two Phase
I/II clinical trials to evaluate the safety of AAV-factor IX gene transfer to
muscle and liver of patients with severe hemophilia B. Herein, we have reviewed
results from studies in animals with hemophilia, early experience with the
vector system in the clinic, recent innovative approaches in vector design and
delivery, and strategies to circumvent immunological limitations. Taken
together, these studies provide much encouragement for the possibility of
future clinical success, but also point out hurdles that still have to be
overcome.
[Back to top] Recent Developments in Recombinant AAV-Mediated Gene Therapy for Lung
Diseases
Terence
R. Flotte
Recent studies
have shed light on a number of important obstacles to safe and effective gene
transfer to the respiratory tract with recombinant AAV vectors. Among these are
blocks at the level of receptor binding and internalizations, evasion of
proteasomal degradation, inefficiency of nuclear entry, and nuclear factors
that inhibit the conversion of rAAV genomes into active double-stranded DNA
form. Other important issues have been the size constraints of the vector, the
lack of retention of episomal forms of the vector genome, and immune responses
which may limit the efficiency of repeated doses of rAAV. Each of these
potential obstacles has been addressed with new vector designs. In addition,
the availability of an abundance of novel rAAV serotypes, each with its own
receptor tropism, has expanded the range of possibilities for long-term success
of gene therapy in the respiratory tract.