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Gene Therapy (2000) 7, 9–13
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MILLENNIUM REVIEW
The application of gene therapy in autoimmune
diseases
CM Seroogy and CG Fathman
Stanford University School of Medicine, Department of Medicine, Division of Immunology and Rheumatology, 300 Pasteur Drive,
Room S021, Stanford, CA 94305-5111, USA
The application of gene therapy in autoimmune disease represents a novel use of this technology. The goal of gene
therapy in the treatment of autoimmune disease is to restore
‘immune homeostasis’ by countering the pro-inflammatory
effects of the CD4+ T cells in the lesions of autoimmunity.
This can be accomplished by adoptive therapy with transduced T cells which can specifically home to the site of
inflammation and secrete ‘regulatory’ protein(s) to ameliorate the inflammation or by direct targeting of the retroviral
vector to activated T cells in the sites of inflammation. Transduction of autoantigen recognizing CD4+ T cells, to secrete
anti-inflammatory products, may become the ‘magic bullet’
to combat the ravages of autoimmune inflammation and
tissue destruction. Gene Therapy (2000) 7, 9–13.
Keywords: gene therapy; autoimmune disease; CD4+ T cell
Introduction
Current human gene therapy clinical trial protocols have
primarily focused on somatic gene replacement and cancer immunotherapy. The application of gene therapy in
autoimmune disease represents a novel use of this technology. Over the past year, clinical trials utilizing gene
therapy for rheumatoid arthritis have begun. The first
trial involved ex vivo infection of synoviocytes with a
retroviral vector and re-injection into a single metacarpophalangeal joint.1 This phase one clinical trial was
designed to test for transgene expression, in this case the
IL-1 receptor antagonist, (IL-1Ra), since the recipients
were scheduled to have joint replacement 1 week after
re-infusion of the transduced synoviocytes. As discussed
below, several laboratories, including our own, have
begun to study the use of targeted gene therapy by using
antigen-specific CD4+ T cells as vehicles for local delivery
of anti-inflammatory proteins in animal models of organspecific autoimmune diseases.
The underlying immunopathogenesis of autoimmune
diseases has not been fully elucidated. The precise role
of B cells, antigen-presenting cells (APC) and T cells in
various autoimmune diseases is unclear. However, CD4+
T cells have been shown to be critically important
mediators of organ-specific autoimmune disease from
studies in animal models,2,3 as well as indirect evidence
in humans.4–6 The CD4+ T cells that appear to be
important in most organ-specific autoimmune diseases
have an effector function consistent with a T helper type
1 (Th 1) polarized cell.7 Several studies have demonstrated the development of Th2-like autoantigen-specific
T cells in the recovery phase of disease, suggesting that
Th2 cytokines such as IL-4 and IL-10 may be important
Correspondence: CM Seroogy
for disease recovery.8–10 This may represent restoration of
a more balanced immune response to auto-antigen stimulation and result in disease amelioration. Although alteration of effector function of a differentiated CD4+ T cell
appears to be unlikely,11,12 it appears to be possible to
modulate the effector function and differentiation of
undifferentiated Th0 T cells and CD4+ T cells that are not
completely differentiated.13 Thus, a desirable approach to
the treatment of autoimmune diseases in humans might
be the selective delivery of regulatory proteins to sites
of inflammation in autoimmune diseases. Several
approaches have been taken utilizing animal models to
provide a rationale for the application of gene therapy in
human autoimmune diseases.
Rationale
Preliminary data in support of the rationale came from
diverse areas. Autoantigen-specific therapies in animal
models have studied the effects of altered peptide ligands
(APLs) and oral tolerance regimens. These studies have
demonstrated that there are important trans-acting effects
at the sites of inflammation that modulate the disease
outcome. Moreover, these effects did not require precise
identification of the autoantigen for therapeutic effect, a
critical point particularly for human disease where the
autoantigen is frequently poorly characterized. For
example, induction of experimental autoimmune
encephalitis (EAE), an animal model of multiple sclerosis,
with myelin basic protein (MBP) Ac1-11 specific T cell
clones can be reversed by treatment with an altered peptide ligand modified for T cells specific for MBP p87-99.14
Studies in vitro with the APL, using MBP p87-99 specific
T cell clones, demonstrated an increase in the ratio of IL4:TNF in favor of a Th2 response. Reverse transcriptase
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CM Seroogy and CG Fathman
10
polymerase chain reaction (RT PCR) of spinal cord tissue
from APL-treated animals revealed no detectable TNF
mRNA, suggesting that the APL might be working by
a similar mechanism in vivo. These authors went on to
demonstrate that the simultaneous treatment of the animal with IL-4 blocking antibodies abrogated the effects
of the APL treatment. These findings provided support
for the concept of gene therapy to replace peptide therapy based on the following. First, the immune modulating effects of IL-4 had a trans-effect: the pathogenic T cell
clones were still present in the lesion of EAE as demonstrated by PCR for T cell receptor (TCR) V gene usage,
but were silenced by the local production of IL-4 and
possibly other unidentified immunomodulatory substances. Second, it was not necessary to use the exact epitope that incited the disease to lead to this ameliorating
effect. Although APL or peptide administration may
seem like a feasible approach for therapy of human autoimmune disease with little inherent risk, there are several
problems. For most human autoimmune diseases, the
autoantigen is not known. In animal models, where most
of these studies have been performed, the disease is typically induced using a particular antigen: eg type II collagen for an animal model of rheumatoid arthritis. It is
more likely that there are multiple autoantigens in
human autoimmune disease. The diseases may start by
an immune response to a particular antigen (either an
autoantigen or a conventional antigen), followed by
breaking tolerance and determinant spreading against
multiple autoantigenic epitopes, thus perpetuating the
immune response.
Oral therapy (with peptides or proteins) has been studied in animal models and in humans with autoimmune
diseases. Important to the concept of gene therapy
approaches to treatment of autoimmune diseases, oral
tolerance experiments in EAE have demonstrated the
feasibility of generating regulatory T cells. Although,
identical to the encephalitogenic CD4+ T cells with
respect to TCR gene usage, MHC allele, and epitope recognition the regulatory T cells differed in their cytokine
production.15 The generation (or targeting) of these regulatory cells appeared to be local to the inflammatory
lesions, since feedings with irrelevant proteins had no
therapeutic effect, despite demonstration of the presence
of regulatory cells for the irrelevant protein. Similar
results have been obtained in other animal models of
autoimmune disease.16–19 The development of these regulatory T cells has been termed active suppression and is
felt to be a vital immune mechanism in peripheral tolerance. Clinical trials are underway using this approach in
human autoimmune diseases. While most studies are
ongoing, completion of a phase II trial of oral myelin
basic protein in multiple sclerosis did not show a significant clinical benefit over the placebo group.20 Although,
a phase two clinical trial (n = 60) studying oral chicken
collagen type II (CII) in severe rheumatoid arthritis demonstrated a significant decrease in joint swelling in
patients receiving CII compared with placebo,21 the
follow-up trial failed to demonstrate efficacy of this
therapy.22
These animal experiments have provided a basis for
rational approaches to T cell-mediated gene therapy in
autoimmune diseases. Transducing an antigen-specific T
cell that could home to the site of inflammation with vectors containing genes that allow expression of regulatory
Gene Therapy
proteins of the type identified in the studies outlined
above, could provide local immunomodulatory effects
that would bypass the problems inherent with systemic
administration of these proteins. T cell-mediated gene
therapy experiments using animal models of autoimmune diseases have been few in number because of
the inability to transduce murine T cells efficiently. In
addition, these experiments have required long incubation periods in vitro, as well as selection methods that
may subsequently lead to alteration in the transduced
T cells.
CD4+ T cells as an approach to targeted
gene therapy
A T cell-based gene therapy approach, which targets sites
of inflammation, could limit the degree of immunosuppression compared with that seen with systemic
immunosuppressive agents. In particular, the local
expression of ‘regulatory proteins’ could be directed to
sites of autoimmune inflammation to delivery regulatory
proteins (ie cytokines or chemokines) with local immunomodulatory effects. This would be a step beyond current
therapeutic modalities. It is unclear what effect various
cytokines will have on disease processes emphasizing the
importance of studies in animal models. One advantage
of gene-modified cells is the ability to insert ‘suicide’
genes into the vector construct allowing the possibility of
killing the gene-modified cells if necessary. This
was recently demonstrated in human trials using the
thymidine kinase gene.23
Currently, the most efficient way to introduce genes
into lymphoid cells is via transduction, utilizing viral vectors. The most widely studied and utilized viral vectors
in humans are murine retrovirus derived. The main
advantages of retroviruses for transduction are chromosomal integration and stable gene expression. Furthermore, these vectors are not associated with inciting an
antiviral inflammatory response, a major consideration
for treatment of autoimmune diseases. The major disadvantages and technical hurdles of retroviral vectors are
two-fold: (1) the cells must be dividing in order to be
transduced; and (2) it is difficult to attain adequate levels
of gene expression. In addition, there is some concern
regarding insertional mutagenesis. However, the data
from human clinical trials, animal studies, and experiments in vitro suggest that the rate of insertional
mutagenesis leading to malignant transformation
appears to be the same or less than the rate of spontaneous mutagenesis.24,25 To date, almost all gene therapy
trials have involved ex vivo infection of cells with reinfusion after extensive safety studies. The retroviral vectors have been modified to become replication-defective
viruses. This ensures that the virus is capable of infecting
only the target cell without further propagation of virus
and resultant infection of other cells following transfer.
This is a critical safety issue, since the presence of replication-competent virus would significantly increase the
risk for insertional mutagenesis. Studies with retroviruses
in vivo have been small in number and have been predominantly performed in animal models. The future of
retroviral gene therapy will involve infusion of modified
retroviruses directly into human tissues avoiding the
expense and time of ex vivo applications. This approach
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CM Seroogy and CG Fathman
is currently hampered by the inability to target the retrovirus selectively to a particular cell, and the requirement
for high-titer virus in order to have an acceptable transduction efficiency.
Transduction of antigen-specific CD4+ T cells in animal
models of autoimmune disease has been hampered by
low transduction efficiencies. Several studies have been
reported, including transduction of T cell hybridomas
from our own laboratory. Collectively, these studies have
demonstrated that transduced antigen-specific T cells can
lead to disease amelioration. One study demonstrated an
ameliorating effect in established disease which has
major implications for treatment in human disease.26 In
these studies, the effect was indirectly demonstrated to
be local or in the environment of autoantigen presentation. Our study utilizing MBP-specific T cell
hybridomas in the animal model EAE, demonstrated a
loss of the ameliorative effects of targeted gene therapy
if T cell receptor (TCR) negative, IL-4 expressing
hybridomas were adoptively transferred.27 This
indirectly suggested that it was not the systemic, but the
local effects of the constitutively expressed IL-4. Furthermore, local production was demonstrated by detection of
IL-4 mRNA in spinal cord tissue of TCR+, IL-4 secreting
hybridomas following successful adoptive gene therapy
in EAE. Systemic levels of IL-4 were not detectable until
well after the initial recovery phase of the disease.
Mathisen et al26 studied the role of IL-10 in preventing
the onset of EAE and treating established disease. In this
study, T cell clones specific for proteolipid protein (PLP)
were transfected in vitro using plasmid DNA and a
polybrene-DMSO transfection method. The murine IL-10
gene was subcloned downstream of the murine IL-2 promoter to allow antigen-inducible, non-constitutive
expression. After selection of the transfectants (requiring
weeks in culture with low transfection efficiency), IL-10
producing cells were intravenously injected into mice
before and after the onset of disease, resulting in protection or amelioration of disease, respectively. This was
inferred to be an antigen-specific response, since adoptive
transfer of an IL-10 expressing T cell clone without CNS
antigen specificity demonstrated no clinical or histopathologic benefit. Confounding this result was the observation that the transfected and adoptively transferred T
cell clone revealed endogenous IL-10 mRNA on an
RNase protection assay, that was the majority of the IL10 message (60% endogenous, 40% transgene). This suggested that the adoptively transferred T cell clone already
had a Th2 phenotype. Previously, co-transfer experiments had established the ameliorating effects of Th2
PLP- or MBP-specific T cells in this disease,28 and since
this experiment did not have a nontransduced T cell
clone control, the results must be interpreted carefully.
Chen et al29 recently described amelioration of EAE with
latent TGF-␤ transduced MBP-specific T cells using a
constitutive retroviral vector. In this experiment, a Th1 T
cell clone was utilized and was not altered with respect
to Th1 mRNA levels after transduction (in this study the
authors had only a 3% transduction efficiency). Furthermore, the nontransduced control had slightly more
severe disease than the immunized control group.
The majority of gene therapy studies using antigenspecific T cells have been done in EAE, one other study
has been reported that was in the neonatal NOD mouse.
Moritani et al30 used a murine retroviral vector containing
the murine IL-10 gene downstream of a constitutive
internal promoter to transduce Th1 islet reactive T cell
clones in vitro. These cells were adoptively transferred to
neonatal NOD mice alone or mixed with unmodified
disease-accelerating Th1 clones. The animals that
received the unmodified Th1 clones had a significantly
higher disease grade (and insulitis) compared with genemodified Th1 clones (66% versus 8%). In addition, by mixing the two populations, the disease incidence was significantly reduced (66% versus 20%) suggesting that this
may have therapeutic benefit in early disease. In this
study, the presence of the neomycin reporter gene in
genomic DNA from recipient islets suggested that the
transferred cells migrated to the pancreas and possibly
proliferated in that area. Notably, this result stands
in contrast to the results published by the same group,
and another group, on transgenic expression of IL-10 in
NOD mice,31,32 illustrating the care needed in interpreting
the implications of various models of potential
immunotherapy.
11
Problems and potential solutions
One major obstacle to T cell-mediated gene therapy studies in animal models of autoimmune disease has been
the inability of investigators to transduce murine T cells
efficiently. Previous studies have utilized T cell
hybridomas or required weeks in culture under selection
conditions that could potentially alter the T cell phenotype. Our laboratory, along with others, has optimized
transduction conditions to allow more meaningful gene
therapy studies.33,34 In these studies, the efficiency of
transduction has been increased up to 60% of the antigenspecific CD4+ T cells allowing the possibility of adoptive
gene therapy with sufficient numbers of recently transduced CD4+ T cells. Another adaptation of these recent
studies has been the incorporation of the marker protein,
green fluorescent protein (GFP), as the basis of selection.
This has allowed a ‘titration’ of the delivered ‘regulatory’
gene product based upon the linear relation demonstrable between GFP expression and the regulatory gene
transcribed through use of an internal ribosomal entry
site (IRES). Additional studies are being planned with a
double IRES vector to allow the insertion of a gene encoding regulatory transcription factors (ie GATA311 or TBet (personal communication L Glimcher)) in addition to
genes encoding the marker and immuno-regulatory proteins. These vectors have been designed to block the
inherent TH1 or TH2 cytokine gene expression of the activated, transduced CD4+ T cells, and allow transgene
regulatory protein to be expressed in the absence
of the counter-regulatory effects of the endogenous
pro-inflammatory cytokines.
Summary
The goal of gene therapy in autoimmune disease treatment is to restore ‘immune homeostasis’ by countering
the pro-inflammatory effects of the CD4+ T cells in the
autoimmune lesions. We hypothesize this could be
accomplished by adoptive therapy with transduced CD4+
T cells which can specifically home to the site of inflammation and secrete their ‘regulatory’ protein(s) to ameliGene Therapy
Millennium review
CM Seroogy and CG Fathman
12
orate the inflammation. Transduction of autoantigen
recognizing CD4+ T cells, which secrete anti-inflammatory products, may become the ‘magic bullet’ to combat
the ravages of autoimmune inflammation and tissue
destruction. Many additional studies need to be performed before this therapy can be applied to human disease.
Can these regulatory gene products, cytokines or chemokines or single chain antibodies, affect ongoing disease?
The answer is unknown but the question must be
addressed before this therapy is applicable to human disease. Is it possible to target the retroviral vector to the
pro-inflammatory T cell in the autoimmune lesion, thus
obviating the need for expensive and potentially dangerous T cell expansion ex vivo? Again, the answer is
unknown but recent advances in ‘re-targeting’ of retroviral vectors might support this possibility.35 It may be
possible to modify the coat protein of the replication
deficient retroviral vector to target the activated CD4+ T
cell through cell surface receptors which uniquely mark
the pathogenic T cell in the lesion and have high affinity
recognition of the modified envelope protein. Thus, the
potential of gene therapy for human autoimmune disease
has been demonstrated in animal models, but caution
must be taken in extrapolating these early successes to
human trials. Cautious optimism may well be the appropriate view of this expanding field. The transduction
efficiency and homing properties of the autoantigen specific CD4+ T cells, as well as the immunologically silent
nature of retroviral transduction should allow rapid
advance in this exciting field of gene therapy for autoimmune disease.
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