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Current
Gene Therapy
ISSN: 1566-5232
Current Gene Therapy
Volume 7, Number 5, October 2007
Contents
Gene Therapy and the Immune System
Guest Editor: Roland W. Herzog
Editorial Pp. 296
Immune Response to Helper Dependent Adenoviral Mediated
Liver Gene Therapy: Challenges and Prospects Pp.
297-305
Michael P. Seiler, Vincenzo Cerullo and Brendan Lee
[Abstract]
Immune Responses to Lentiviral Vectors Pp.
306-315
Antonia Follenzi, Laura Santambrogio and Andrea Annoni
[Abstract]
Immune Responses to AAV in clinical trials
Pp. 316-324
Federico Mingozzi and Katherine A High
[Abstract]
AAV as An Immunogen Pp. 325-333
Luk H. Vandenberghe and James M. Wilson
[Abstract]
Immune Responses to Gene Product of Inducible Promoters
Pp. 334-346
Caroline Le Guiner, Knut Stieger, Richard O. Snyder, Fabienne
Rolling and Philippe Moullier
[Abstract]
Immune Responses to Adenovirus and Adeno-Associated
Vectors Used for Gene Therapy of Brain Diseases: The Role
of Immunological Synapses in Understanding the Cell Biology
of Neuroimmune Interactions Pp. 347-360
Pedro R. Lowenstein, Ronald J. Mandel, Wei-dong Xiong,
Kurt Kroeger and Maria G. Castro
[Abstract]
Immune Responses to Gene-Modified T Cells
Pp. 361-368
Linda Mesler Muul and Fabio Candotti
[Abstract]
Tolerance Induction by Gene Transfer to Lymphocytes
Pp. 369-380
Jonathan Skupsky, Yan Su, Tie-Chi Lei and David W. Scott
[Abstract]
Emerging Role of Regulatory T Cells in Gene Transfer
Pp. 381-390
Ou Cao, Christian Furlan-Freguia, Valder R. Arruda and
Roland W. Herzog
[Abstract]
Recent Advances in Immune Modulation Pp.
391-402
Carol H. Miao
[Abstract]
Immunology of Neonatal Gene Transfer Pp.
403-410
Katherine P. Ponder
[Abstract]
Abstracts
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Editorial : Gene Therapy and the
Immune System
While based on a simple concept of introducing a functional
gene to replace a non-functional sequence of a person’s
genome or to otherwise add a therapeutically beneficial gene,
gene therapy is faced with a complex set of interactions between
the gene transfer vector, the transgene, and the recipient
of gene transfer. Following tremendous advances in vector
development, cures have been reported in numerous animal models
of human diseases, and gene therapy now holds much promise
as a novel form of molecular medicine. However, complications
of treatment related to insertional mutagenesis following
integration of vector DNA into host chromosomes and to untoward
immune responses against vectors and transgene products have
merged as serious obstacles for successful translation to
humans. This issue of Current Gene Therapy is dedicated
to an in-depth assessment of immune responses in gene transfer.
The importance of the topic is further highlighted by the
recent meeting of the NIH’s Recombinant DNA Advisory
Committee (RAC), which discussed vector-specific T cell responses
in a gene therapy trial for hemophilia on 19 June 2007, and
by reports on complications of gene therapy for severe inflammatory
disease.
The immune system has evolved to differentiate between self
and foreign molecular structures and sequences and to respond
to activation signals that indicate danger to the body. Consequently,
the immune system may fight off “invading” gene
transfer vectors or foreign protein and nucleic acid sequences.
Innate immunity provides a rapid defense system that responds
to exogenous signals (e.g. pathogen-associated molecular patterns
such as bacterial or viral nucleic acids) and to endogenous
signals that, for example, may be derived from tissue damage
during vector administration, cellular stress, or viral infection.
The adaptive immune response is delayed but more specific
(involving presentation of antigen to highly specific T and
B cell receptors) and also generated memory B and T cells.
Cytotoxic T cell and antibody responses may block gene transfer
or eliminate therapeutic gene expression. The articles in
this issue reflect that the potential for any of these immune
responses in gene transfer depends on many factors, including
the gene transfer vector (type of vector, specifics of the
construct such as promoters, envelope/capsid, purity, etc.),
vector dose, route of administration (ex vivo, in vivo,
target organ, method of delivery), age and genetic factors
of the recipient of gene therapy, and the nature of the transgene
product (self, non-self, cellular localization, etc.).
Nonetheless, research is well under way to overcome these
hurdles. We have just begun to understand the interactions
between vectors and the immune system of specific target tissues
(organ-specific immunity). For example, recent research demonstrates
the importance of interactions with bone marrow-derived professional
antigen presenting cells. Similar to research in transplantation
biology, gene therapists have uncovered strategies to manipulate
the immune system, leading to sustained transgene expression
in animal models. At the same time, we can take advantage
of mechanisms of immune tolerance and regulation mechanisms,
which have evolved to prevent autoimmunity and destructive
immune responses.
Roland W. Herzog
Associate Editor
University of Florida
Cancer and Genetics Research Center
1376 Mowry Road, Room 203
Gainesville, FL 32610
USA
Phone: 352-273-8113
Fax: 352-273-8342
E-mail: rherzog@ufl.edu
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Immune Response to Helper Dependent Adenoviral
Mediated Liver Gene Therapy: Challenges and Prospects
Michael P. Seiler, Vincenzo Cerullo and Brendan Lee
Adenovirus-mediated gene therapy holds significant potential
especially for applications requiring high levels of target
tissue transduction. While significant advances in clinical
adenoviral gene therapy applications have been made in cancer,
the clinical translation of adenoviral gene replacement therapy
for genetic disease has lagged. Encouragingly, advances in
vector production have led to the development of Helper-Dependent
(“gutted” or “high capacity”) adenoviral
vectors (HDV) deleted of all viral coding genes. HDV significantly
reduces the chronic toxicity associated with early generation
adenoviral vectors that has been most significant after systemic
administration in both small and large animal models. However,
the field remains confounded by innate immune responses inherent
to adenovirus, and more generally, to the adaptive immune
response to transgene. Together they decrease the effective
therapeutic index for any particular treatment. This review
summarizes the current advances toward understanding the decisive
cell and molecular mechanisms underlying the acute toxicity
to systemic HDV administration. We focus on the complex immune
response and consequences of systemic vector delivery in the
context of liver-directed monogenic disease therapy. Future
development of interventions to avoid the innate immune response,
including vector and pharmacologic manipulations, should further
contribute to minimizing vector toxicity while maximizing
the efficacy of systemic HDV gene transfer.
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Immune Responses to Lentiviral Vectors
Antonia Follenzi, Laura Santambrogio and Andrea Annoni
Efficient delivery and sustained expression of a therapeutic
gene into human tissues are the requisite to accomplish the
high expectations of gene therapy. A major challenge has concerned
development of gene transfer systems capable of efficient
cell transduction and transgene expression without harm to
the recipient. A lot of work has been done to demonstrate
the efficacy of gene therapy in animal models that mimic situations
in humans. Use of lentiviral vectors (LVs) offers multiple
advantages for gene replacement therapy, because they combine
efficient delivery, ability to transduce proliferating and
resting cells, capacity to integrate into the host chromatin
to provide stable long-term expression of the transgene, absence
of any viral genes in the vector and absence of interference
from preexisting viral immunity. However, one of the major
barriers to stable gene transfer by LVs and other viral vectors
is the development of innate and adaptive immune responses
to the delivery vector and the transferred therapeutic transgene.
Since this greatly hinders the therapeutical benefits of gene
therapy by LVs, developing strategies to overcome the host
immune response to the transfer vector and the transgene is
a matter of current investigation.
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Immune Responses to AAV in clinical trials
Federico Mingozzi and Katherine A High
Recent findings in a clinical trial in which an adeno-associated
virus (AAV) vector expressing coagulation factor IX (F.IX)
was introduced into the liver of hemophilia B subjects highlighted
a new issue previously not identified in animal studies. Upon
AAV gene transfer to liver, two subjects enrolled in this
trial developed transient elevation of liver enzymes, likely
as a consequence of immune rejection of transduced hepatocytes
mediated by AAV capsid-specific CD8+
T cells. Studies in healthy donors showed that humans carry
a population of antigen-specific memory CD8+
T cells probably arising from wild-type AAV infections. The
hypothesis formulated here is that these cells expanded upon
re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer,
and cleared AAV epitope-bearing transduced hepatocytes. Other
hypotheses have been formulated which include specific receptor-binding
properties of AAV-2 capsid, presence of capsid-expressing
DNA in AAV vector preparations, and expression of alternative
reading frames from the transgene. Absence of a valid animal
model has prevented an in-depth mechanistic study of the phenomenon.
Several possible solutions to the problem are discussed, including
the administration of a short-term anti-T cell immunosuppression
regimen concomitant with gene transfer. While more studies
will be necessary to further define mechanisms and risks associated
with capsid-specific immune responses in humans, monitoring
of these responses in clinical trials will be essential to
achieving the goal of long-term therapeutic gene transfer
in humans.
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AAV as An Immunogen
Luk H. Vandenberghe and James M. Wilson
The first in vivo adeno-associated viral vector (AAV)
gene transfer experiments were performed in murine models
of muscle directed gene transfer. These studies were remarkable
for stable expression of a variety of immunogenic transgenes.
These findings were translated to other target organs with
multiple therapeutic gene products. Technological improvements
and the lessons learned from basic research have heralded
an era of first-in-human clinical trials. In most settings,
AAV appears to evade host immune surveillance, allowing the
delivery of robust levels of genetic cargo that leads to persistent
expression. However, in few experimental settings immunological
responses raised following AAV mediated gene transfer have
compromised vector efficacy. Parameters that determine these
occurrences have been proposed to be pre-existing immunity
to AAV, the route of administration, the kinetics of expression,
the dose, the vector serotype and its ability to transduce
antigen-presenting cells (APCs) as well as the host species
and nature of the specific transgene product. Overall, the
underlying mechanisms remain the topic of scientific debate.
This review aims to compile, confront and critically discuss
the findings in which AAV appears to be an immunogen.
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Immune Responses to Gene Product of Inducible Promoters
Caroline Le Guiner, Knut Stieger, Richard O. Snyder, Fabienne
Rolling and Philippe Moullier
Efficient gene transfer has been achieved in several animal
models using different vector systems, leading to stable transgene
expression. The tight control of this expression is now an
important outcome for the field of gene therapy. Such regulation
is likely to be required for therapeutic applications and
in some instances for safety reasons. For this purpose, several
regulatable systems depending on small molecules have been
developed. Among these, the tetracycline and the rapamycin
dependent systems have been largely used. However, if long-term
regulation of the transgene has been obtained in small animal
models using these inducible systems, when translational studies
were initiated in larger animals, the development of an immune
response against proteins involved in transgene regulation
were often observed. Such immune response was especially documented
when using the TetOn tetracycline regulatable system in nonhuman
primates (NHP). Humoral and destructive cellular immune responses
against the transactivator involved in this regulation system
were documented in a large majority of NHP leading to the
complete loss of the transgene regulation and expression.
This review will describe the immune responses observed in
these different model systems applied for transgene regulation.
Focus will be finally given on future directions in which
such immune responses might be surmounted, enabling long-term
transgene regulation in future clinical developments of gene
transfer.
[Back to top]
Immune Responses to Adenovirus and Adeno-Associated
Vectors Used for Gene Therapy of Brain Diseases: The Role
of Immunological Synapses in Understanding the Cell Biology
of Neuroimmune Interactions
Pedro R. Lowenstein, Ronald J. Mandel, Wei-dong Xiong,
Kurt Kroeger and Maria G. Castro
Researchers have conducted numerous pre-clinical and clinical
gene transfer studies using recombinant viral vectors derived
from a wide range of pathogenic viruses such as adenovirus,
adeno-associated virus, and lentivirus. As viral vectors are
derived from pathogenic viruses, they have an inherent ability
to induce a vector specific immune response when used in
vivo. The role of the immune response against the viral
vector has been implicated in the inconsistent and unpredictable
translation of pre-clinical success into therapeutic efficacy
in human clinical trials using gene therapy to treat neurological
disorders. Herein we thoroughly examine the effects of the
innate and adaptive immune responses on therapeutic gene expression
mediated by adenoviral, AAV, and lentiviral vectors systems
in both pre-clinical and clinical experiments. Furthermore,
the immune responses against gene therapy vectors and the
resulting loss of therapeutic gene expression are examined
in the context of the architecture and neuroanatomy of the
brain immune system. The chapter closes with a discussion
of the relationship between the elimination of transgene expression
and the in vivo immunological synapses between immune
cells and target virally infected brain cells. Importantly,
although systemic immune responses against viral vectors injected
systemically has thought to be deleterious in a number of
trials, results from brain gene therapy clinical trials do
not support this general conclusion suggesting brain gene
therapy may be safer from an immunological standpoint.
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Immune Responses to Gene-Modified T Cells
Linda Mesler Muul and Fabio Candotti
Gene-modified T cells were the first gene therapy tool used
in clinical gene transfer trials. After the first applications
in immunodeficiency diseases, T cell gene therapy has been
extended to HIV infection and cancer. The primary obstacle
to successful T cell gene therapy has proven to be the robust
immune responses elicited by the gene-modified T cells even
in severely immunosuppressed patients. The potent antibody
and cytotoxic immune responses have interfered with the expression
and persistence of the therapeutic transgene. In this review
we will address each of the components of T cell gene therapy
– culture conditions, vector, and transgene –
that have elicited these immune responses and the strategies
used to minimize them.
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Tolerance Induction by Gene Transfer to Lymphocytes
Jonathan Skupsky, Yan Su, Tie-Chi Lei and David W. Scott
Tolerance must be maintained to prevent deleterious immune
responses. Thus, when tolerance is lost, autoimmunity can
result. A number of novel approaches to (re-) induce tolerance
for potential clinical applications have been developed in
the last decade. Our lab has implemented an immunoglobulin-based
gene therapy approach, which may have powerful implications
for the treatment of human conditions. These include a variety
of autoimmune diseases, transplantation, and the immune response
to therapeutic proteins (as in the treatment of hemophilia
A) or gene therapy per se. We clone the target (immunogenic)
protein in frame with an immunoglobulin heavy chain and deliver
it via retrovirus to an activated B cell. In our system, we
observe tolerance to multiple epitopes of the protein cloned.
An important advantage of this regimen is that identification
of the precise peptide epitopes of a target protein is not
necessary since selection and presentation by the host’s
own antigen presenting cells (APC’s) eliminates the
issue of HLA polymorphism. Additionally, our data indicate
that these tolerogenic B cells are stimulating an endogenous
population of regulatory T cells, which are effective at suppressing
the immune response in both naïve and primed hosts. Thus,
this approach has potential for future clinical therapy.
[Back to top]
Emerging Role of Regulatory T Cells in Gene Transfer
Ou Cao, Christian Furlan-Freguia, Valder R. Arruda and
Roland W. Herzog
Induction and maintenance of immune tolerance to therapeutic
transgene products are key requirements for successful gene
replacement therapies. Gene transfer may also be used to specifically
induce immune tolerance and thereby augment other types of
therapies. Similarly, gene therapies for treatment of autoimmune
diseases are being developed in order to restore tolerance
to self-antigens. Regulatory T cells have emerged as key players
in many aspects of immune tolerance, and a rapidly increasing
body of work documents induction and/or activation of regulatory
T cells by gene transfer. Regulatory T cells may suppress
antibody formation and cytotoxic T cell responses and may
be critical for immune tolerance to therapeutic proteins.
In this regard, CD4+CD25+
regulatory T cells have been identified as important components
of tolerance in several gene transfer protocols, including
hepatic in vivo gene transfer. Augmentation of regulatory
T cell responses should be a promising new tool to achieve
tolerance and avoid immune-mediated rejection of gene therapy.
During the past decade, it has become obvious that immune
regulation is an important and integral component of tolerance
to self-antigens and of many forms of induced tolerance. Gene
therapy can only be successful if the immune system does not
reject the therapeutic transgene product. Recent studies provide
a rapidly growing body of evidence that regulatory T cells
(Treg) are involved and often
play a crucial role in tolerance to proteins expressed by
means of gene transfer. This review seeks to provide an overview
of these data and their implications for gene therapy.
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Recent Advances in Immune Modulation
Carol H. Miao
Successful gene therapy protocols rely on the hypo-responsiveness
of the immune system to transgene products generated from
gene transfer vectors. In order to prevent cytotoxic lymphocyte
or antibody formation induced by transgene expression, various
strategies derived from recent advances in immune tolerance
induction protocols have been tested in gene therapy model
systems. Current immunosuppressive drugs were used to nonspecifically
target T-cell activation, clonal expansion, and differentiation
into effector cells. Central tolerance can be induced from
intrathymic deletion of T cells with thymically expressed
antigens or generation of hematopoietic mixed chimerism. Peripheral
tolerance to transgenes may be achieved by several different
pathways including deletion of activated/effector T cells
by depleting antibodies, generation of T cell apoptosis or
anergy by costimulation blockade, and active suppression by
T regulatory cells. This review outlines the development of
these strategies using various immune modulation regimens
and protocols to induce long-term immune tolerance specific
to the transgene product.
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Immunology of Neonatal Gene Transfer
Katherine P. Ponder
Gene therapy could result in the permanent correction
or amelioration of the clinical manifestations of many genetic
diseases. However, immune responses to the therapeutic protein
pose a significant hurdle for successful gene therapy. Problematic
immune responses can include the development of a cytotoxic
T lymphocyte (CTL) response that results in the destruction
of genetically-modified cells and/or the formation of antibodies
directed against the therapeutic protein. One approach to
avoid an immune response is to perform gene therapy in newborns,
which takes advantage of the fact that the immune system is
relatively immature at birth. This approach has been highly
effective in mice, and has resulted in stable expression without
antibody formation for proteins that are highly immunogenic
after transfer to adults. High levels of expression after
neonatal gene therapy were more effective at inducing tolerance
than low levels of expression in mice, which suggests that
high antigen levels are more efficient at inducing tolerance.
A criticism of this approach is that the murine immune system
is less mature at birth than the immune systems of larger
animals. Indeed, neonatal gene therapy to cats with mucopolysaccharidosis
I resulted in a CTL response that destroyed expressing cells.
Nevertheless, the immune system was still relatively immature,
as transient administration of a single immunosuppressive
agent at the time of neonatal gene therapy resulted in stable
expression. Neonatal administration can reduce, but not eliminate,
immune responses after gene therapy.
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