Cells. Tissue source(s). Marker gene. Disease state. Investigator. Autologous. WBM neo ... (University of. Indiana) ..... of Alabama). I.I)1. .... Mechanisiti lnvestigaior. HIV-lT(v). Auiologous fibroblasts. Vaccine. Galpin. (USC). revM .... Tompkins,.
REVIEW
Gene
therapy: William
current
G. Kerr
Department
status
and James
of Gene
Alto,
Palo
Ca4fornia
Abstract: The ability to successfully introduce foreign genes into eukaryotic cells has made possible a new approach to the treatment of human disease. Gene therapy is now being brought to bear on genetic, malignant, and infectious diseases. In this review, we summarize the status of the field through an analysis of clinical volving transfer of marker or therapeutic various somatic cell targets. Many of these gin to examine whether or not patients with are unresponsive to conventional therapeutic
protocols ingene(s) into trials will bediseases that interven-
tions (e.g., pharmaceutical, surgical) will show benefit solely from somatic cell-based gene therapies. In other trials, the gene therapies are meant to complement or bolster the effect of conventional treatments. Although the field will
of gene therapy is now entering the clinical arena, also attempt to summarize certain shortcomings
technical eventual 1994.
hurdles widespread
Key Words: disease A IDS
that
retrovirus
will use.
J.
need to Leukoc.
. chemotherapy
. infrctious
be overcome Biol. 56:
tumor
. expression
disease
we and
for 210-214;
immunity factors
its
. genetic
INTRODUCTION Gene therapy mammalian
is the introduction somatic cells in order
of genetic material to treat malignant,
into infec-
tious, or inborn genetic diseases. This relatively new field is poised to become a significant addition to the armamentarium of the clinician faced with curing disease. It is clear that the field of gene therapy has reached a watershed - the efficacy of gene therapy for the treatment of human disease will finally be tested. In this review we present a concise summary of all gene therapy clinical trials, as approved by the National Institutes of Health Recombinant DNA Advisory Committee (RAC). Such a survey is particularly poignant at this defining moment in the development of gene therapy technology. For reviews of experimental studies of gene thenapy
we
Gene
refer
the
Delivery
reader
elsewhere
210
and
generation
Journal
Systems
quences for this cis-acting quences
inside the target cell, and DNA copy of the virus cell. There are specific,
(4) integration into the chrononcoding se-
in the retroviral genome that must be present in cis sequence of events to take place. In addition to these sequences, there are certain retroviral coding Sewhose protein products (gag, pol, env) must be
provided in trans for this sequence of events to occur. These coding sequences are expressed in the packaging cell line in such a manner that their protein products can be packaged into virions while their mRNAs are incapable ofbeing packaged into the virions. Thus, providing the necessary components for the packaging and transmission of the recombinant viral genome, but not for the coding sequences of the wild type, results in transduced target cells that are unable to susviral replication, preventing the spread of the virus to other cells. This feature of retroviral systems provides a safe and efficient means of gene transfer. Other virus-based gene transfer technologies exist and are based on the use of recombinant forms of the following viruses: adenovirus, adeno-associated virus (AAV), herpesvirus, vaccinia virus, poliovirus, Sindbis, and RNA viruses [1]. Thus far, only adenoviral systems have seen significant application in the clinical setting. Adenoviral gene delivery systems are based on the demonstration that adenovirus genomes with deletions in the El region can be propagated in cells that express the El genes [12]. Subsequently, it was shown that replication-defective adenovirus genomes harboring foreign DNA sequences could be replicated in cells in which El proteins are supplied in trans, ushering in the use of these vectors in gene therapy applications [5, 6]. tam
Physical begun from
methods
for
to be explored; the drawback
the
delivery
however, that they
of DNA
into
cells
some of these systems lead only to transient
have
suffer expres-
sion of the delivered gene. These physical lipofection, ligand-DNA conjugates, DNA conjugates, direct injection of DNA complexes, and CaPO4 precipitation [1].
methods include adenovirus-ligand or liposome-DNA
GENE
CELL
MARKING
OF
THERAPEUTIC
POPULATIONS
of a double-stranded
of Leukocyte
viral genomic RNA of the double-stranded matin of the target
[ 1-4].
Although there exist several different approaches for transferring genes into mammalian cells [5-11], the majority of clinical gene therapy trials to date employ retroviral vectors as the delivery strategy of choice. Retroviral vector production systems have been developed that permit recombinant retroviral genomes to be transmitted from a viral packaging cell line to a target cell in the absence of wild-type virus [1]. To accomplish this feat, the recombinant viral genome has been engineered to contain only sequences that are necessary for (1) production of the viral genomic RNA, (2) packaging ofthe viral genomic RNA into a virion, (3) reverse transcription
prospects
J. Mule
5)Stemix,
Therapy,
and future
Biology
DNA
Volume
copy
56,
August
of the
1994
Tumor-infiltrating
lymphocytes
Trials are ongoing to determine, capacity of tumor-infiltrating
Abbreviations: simplex
repeat;
TIL,
Reprint mix,
HIV,
virus
3155
Received
human
thymidine
immunodeficiency
kinase;
IL-2,
tumor-infiltrating requests: Porter
April
4,
G. Palo
1994;
Kerr,
Alto,
accepted
TNF,
Department CA
April
gene marking, (TILs) to
virus;
interleukin-2;
lymphocyte;
William Drive,
by stable lymphocytes
94304.
4,
1994.
HSV-TK, LTR,
the traffic
herpes
long
terminal
tumor
necrosis
factor.
of Gene
Therapy,
SySte-
1.
TABLE Tumor
Clinical
Marker
target
Melanoma Ovarian Renal carcinoma5 Melanoma
5Will
Inv olving
Gene
Marking
gene
ease cells in the recipient that survived high-dose chemotherapy. A caveat with this strategy, however, is that if tumor relapse demonstrates a lack of genetic marking it does not formally rule out a tumor-contaminated transplant. Because of their moderate to low transduction efficiency, current retroviral vectors may not adequately transduce tumor cells present at very low frequency in the transplant. Brenner and colleagues [15] have found gene-marked tumor cells contributing to relapse.
of TILs
Investigator
neo neo neo neo neo
Melanoma5
‘ Pending
Trials
Rosenberg
(NIH)
Freedman
(Tulane)
Economou
(UCLA)
Economou (UCLA) Lotze (University
of Pittsburgh)
approval. utilize
exclusively
CD8
TILs
(depleted
of
the
CD4
subset).
THERAPEUTIC to tumor
deposits
as well
as to persist
long-term
in cancer
Cancer
pa-
tients. TILs are T lymphocytes that have localized in tumor masses and are subsequently expanded ex vivo in interleukin-2 (IL-2) [13]. Upon adoptive transfer in animals and in patients with certain tumors, TILs mediate tumor regression [13, 14]. For some tumor histologies, TILs have unique tumor specificity as measured by cytolytic activity and/or secretion of cytokines. In the gene marking studies, TILs are transduced ex vivo with a retrovirus encoding a ne-
Gene
progenitor/stem
TRIALS
therapies therapies
to
combat
cancer
fall
into
three
general
categories: those designed to (1) boost or create immunity to cancer cells (e.g., production of cytokine); (2) specifically poison or prevent proliferation of cancer cells (e.g. , suicide genes, antisense oligonucleotides); or (3) increase the number of patients chemotherapy apeutic agents hematopoietic
omycin resistance gene (neo) and expanded in IL-2 using selection media containing the neomycin analogue G418 before adoptive transfer. Trials involving gene marking are summarized in Table 1.
Hematopoietic
GENE
Most way
of the therapeutic trials for cancer currently under introducing one of several cytokine genes into tumor cells or autologous fibroblasts. The rationale for trials is that, when reintroduced, these genetically
involve
either
these
cells
who can potentially benefit from high-dose as well as increase the amount of chemotheradministered to a patient by protecting the system (chemotherapy resistance genes).
modified
cells
will
serve
as sites
of cytokine
production
and (de-
In the clinical studies outlined in Table 2 the target population being marked consists of hematopoietic stem/progenitor cells. In these protocols, autologous bone marrow or mobilized peripheral blood cells (containing progenitor/stem cells) are reinfused into patients as a rescue from myelosuppression/ablation following administration of high-dose chemotherapy. Because hematopoietic stem cells and early progenitors represent only a small fraction of the mononuclear cells in bone marrow or mobilized peripheral blood, some investigators have chosen to employ CD34-enriched cells. The primary aim of these studies is to determine, in cases
thus
of disease recurrence, whether tumor cells that may contaminate the transplanted bone marrow or mobilized peripheral blood contributed to the relapse. Because these particular transplants are autologous, only genetic marking of the transferred cells offers a reliable method for determining whether disease relapse is due to contaminating tumor cells reintroduced with the transplant or due to residual dis-
A third approach that is currently being taken in a clinical setting is the introduction of suicide genes directly into tumors; the gene ofchoice to date is the herpes simplex virus thymidine kinase (HSV-TK) gene. Expression of this gene renders cells susceptible to killing by ganciclovir. This drug is nontoxic to nonmodified human cells but kills cells carrying the HSV-TK gene by converting the enzyme into
TABLE
Tissue
source(s) WBM
Autologous
WBM
Autologous
WBM
Autologous Autologous
WBM CD34’
Autologous
CD34
Autologous
CD34
Autologous
CD34 CD34
Autologous
CD34
Autologous
CD34’
Autologous
CD34’
Autologous
CD34
Autologous
WBM
‘Will
‘In
compare
identical
Clinical
Marker
Autologous
Autologous
2.
Trials
Involving
Gene
cells
isolated
from
gene
both
immunity
by
several
of
Hematopoietic
Disease
Relapse
Progenitor/Stem
Cells
Investigator
state
Brenner
(Si.
Brenner
(St.
CML ALL CML
Deisseroth
AML
Cornetta
myeloma cancer
CML Breast
and
or
Hodgkin’s
Jude’s) Jude’s) (M.D.
Anderson)
(University
of
(ML).
Deisseroth
Breast
blood
mechanisms
Neuroblastoma
Multiple
peripheral
different
pending on the cytokine of choice) [2]. Another way to induce immunity to tumor cells via gene therapy is to augment the antigenicity of tumor cells. This approach has been taken by Nabel and colleagues by introducing the HLA-B7 gene into malignant melanomas by either retroviral vectors or by DNA/liposome complexes [16]. Tumor cells that express the foreign HLA molecule should trigger a vigorous allogeneic immune response at the tumor site resulting in a coincidental immune response to specific tumor antigens expressed by the malignant cells [2].
Marking
neo neo neo neo neo neo neo neo neo neo neo neo neo neo CD34
enhance
Dunbar
(NIH)
Dunbar
(NIH)
Dunbar
(NIH)
Scheuning
(FHCRC)
Lymphoid
malignancy
Scheuning
(FHCRC)
Lymphoid
malignancy’
Scheuning
(FHCRC)
Leukemia/lymphoma
I)eisseroth
(M
CLL AML
Deisseroth
(M.D.
bone
Brenner
(St.
.
Indiana)
Anderson)
D.
Anderson) Anderson)
Jude’s)
marrow.
twins.
Kerr
and
Mute
Gene
therapy:
current
status
and
future
prospects
211
nucleotide-like precursors that inhibit DNA synthesis. Because retroviruses are capable of selectively transducing dividing cells, the preferential delivery of the HSV-TK gene to tumor masses can be accomplished by stereotaxic injection of retroviral supernatant or producer cells. It is believed that the only significant source of dividing cells in the brain would be of tumor origin. The patients are then treated with ganciclovir to eradicate tumor cells. Interestingly, cated that not all tumor
selectively HSV-TK-expressing experiments in animals have mdicells need express HSVTK for this
approach to eradicate the tumor mass completely. This “bystander” effect is not well understood, but one of several mechanistic theories proposed involves the ability of toxic metabolites to cross gap junctions between neighboring cells within the tumor [17]. A rather similar strategy, but utilizing different therapeutic genes, is the selective induction of antiproliferative effects in tumor cells by the introduction and expression of genes encoding antisense oligonucleotides (Table 3). As already mentioned, another approach to gene therapy
Genetic
disease
therapies
Gene therapies are now being implemented clinically for diseases resulting from recessive single-gene defects. Presumably these disorders would be cured ifa functional copy of the mutated gene was expressed in the appropriate target tissue. However, in disorders in which the gene product is secreted, such as Gaucher’s disease and clotting factor deficiencies, several somatic cell types could conceivably be the target for gene transfer. In others, the functional gene must be delivered and expressed in the appropriate cell type; for example, in thalassemia the 13-globin gene must be delivered and expressed in cells of the erythroid lineage. Five diseases resulting from genetic mutations are currently the focus of NIH RAC-approved clinical trials: severe combined immune deficiency (SCID) syndrome, disease, Fanconia’s anemia (C), cystic fibrosis, cholesterolemia. In Table 4, we list the genes matic cells targeted by these trials.
Gaucher’s and hyperand the so-
for cancer is to protect a given patient’s hematopoietic systern from the toxic effects of high-dose chemotherapy. The chemotherapy resistance gene currently being tested in clinical gene therapy trials is MDR-1. This gene encodes a protein (P glycoprotein) that is capable of serving as a multidrug effiux pump to remove from a cell certain chemotherapeutic agents (e.g., taxol). However, the MDR-1 pump is not effective against most alkylating agents, which are used in many high-dose chemotherapy regimes, thus limiting the usefulness ofMDR-1 gene therapy to specific chemotherapy applications. Additional genes to endow cells of hematopoietic lineages with selective resistance to chemotherapeutic agents and radiation are under consideration for future clinical
AIDS is currently the only infectious disease for which there are approved clinical gene therapy trials (Table 5). The strategies for treatment ofthis disease are to protect susceptible cells from infection by human immunodeficiency virus (HIV) or to inhibit further replication of HIV in already infected cells. These approaches are referred to as intracellular immunization, a term coined by Baltimore [18] in a cornmentary on a research article by McKnight and co-workers [19]. McKnight and co-workers showed that cells stably expressing a truncated form of the herpes simplex virus (HSV)
gene
transactivator
therapy
applications.
Therapeutic
gene
TABLE
3.
Target
cell
Gansbacher
Melanoma
Lotze
tumor
Melanoma
Chang
Autologous
tumor
Neuroblastoma
Brenner
Autologous
tumor
Renal
Simons
Autologous Autologous
tumor tumor
Melanoma SCLC
Seigler Cassileth
Autologous
IL-2 GM-CSF IFN--y IL-2 IL-2 HLA-B7 HSV-TK HSV-TK HSV-TK HSV-TK HSV-TK HSV-TK
212
Journal
into
tumor
of Leukocyte
via
and
tumor
Allogeneic
tumor
Melanoma
DasGupta
Allogeneic
tumor
Melanoma
Sznol
Penitoneal
transfer
Investigator
Renal
fibroblasts
gene
Cancer
(NIH)
Autologous
metastasis
(M-SKI) (M-SKI) (University
of
(University (St.
Pittsburgh)
of
Michigan)
Jude’s)
(Johns
Hopkins)
(Duke University) (University of Miami) (University
of
Ovarian
Freeman
site site site site site
in in in in in
brain brain brain brain brain
Brain Brain Brain Astrocytoma Leptomingeal
Oldfield (NIH) Culver (Methodist, Kun (St. Jude’s) Raffel/Culver (USC) Oldfield/Ram (NIH)
Tumor
site
in
lungs
NSCLC
Roth
Tumor Tumor CD34 CD34 CD34
site in brain sites in liver
Brain Colorectal
Ilan (Case Western Reserve) Rubin (Mayo Clinic)
cells
Ovarian
hematopoietic
cells
Ovarian
hematopoietic
cells
Breast
Metastasis
Melanoma
Metastasis
Solid
DNA-liposome
Biology
Volume
complex.
56,
August
1994
carcinomatosis
adenocarcinoma and
breast
(Tulane)
(M.D.
Iowa)
Anderson)
Deisseroth
(M.D.
Hesdorifer
(Columbia
O’Shaughnessy
tumors
Illinois)
(NIH)
Tumor Tumor Tumor Tumor Tumor
hematopoietic
of
of Cancer
Gansbacher
tumor
‘Direct
replication
Rosenberg
Allogeneic
132-microglobulin’
support
Melanoma
IL-2 IL-4 IL-4
and
not
Melanoma
tumor
(IGF-l)
did
tumor
Allogeneic
Autologous
Antisense HLA-B7’ MDR-l MDR-1 MDR-1 HLA-B7’ HLA-B7
Therapy
VP16
(NIH) (NIH)
IL-2
k-RAS)
protein
Rosenberg Rosenberg
Autologous
and
Gene
therapies
Melanoma Melanoma
TIL
TNF IL-2
(P53
for
disease
tumor
TNF
Antisense
Trials
Infectious
Anderson) University)
(NIH)
Nabel
(University
of
Nabel
(University
of Michigan)
Michigan)
TABLE
1 ttiraput
n
larget
ttflC
ADA AI)A
CI)34 CI)34
cells/autologous cells/PBL
Glucocerebrosidase
CD34
Glucocerrbrosidase
CD34’
Glucocerebrosidase
(Il/i
4.
Trials
cit Gene
lherapy
for
(enetic
ISSUC
cord
blood
Genetic
Disease
disease
Investigator
SCII) SCIL)
Blaese Blaese
cells/PBL
Gaucher’s
Karlsson
cells/PBL
Gaucher’s
Barranger
(University
CD34
cells/PBL
Gaucher’s
Scheuning
(FHCRC)
FACC
CD34’
cells/PBL
CFTR
Airway
Fanconia’s
anemia
Cystic
fibrosis
(C)
Liu
(NIH) (NIH) (NIH)
and
Crystal
Young
of
Pittsburgh)
(NIH)
(NIH)
CFTR’
Airway
Cystic
fibrosis
Wilson
CF1’R
Airway
Cystic
fibrosis
Welsh
CF]R’
Airway
Cystic
fibrosis
Wilmott
(Children’s,
Cincinatti)
CFTR’
Airway
Cystic
fibrosis
Boucher
(University
of
CFTR’
Airway
Cystic
fibrosis
Sorscher
and
(University
Hepatocytes
Hypercholesterolemia
Wilson
(University
I.I)1. .‘
receptor
These
trials
Gene
delivery
will
utilize via
cationic
a replication
defective
adenovirus
vector
to
transduce
the presence the function
ofthe truncated of the wild-type
stem cells efficiently cx vivo without simultaneously driving these cells to commitment to one or more of the hematolymphoid lineages, retroviral transduction would most likely become a more efficient means of introducing relevant genes into these cells for wider therapeutic application. As the field of gene therapy matures, it is becoming apparent that improvements must also occur in expression of the introduced gene. Research into cellular enhancer and
‘IABLE
larg(t
g(it(
HIV-lT(v)
Auiologous
revM
Peripheral
Hy-l’K
Autologous
anti-5-UlS
HIV
ribozvme
IlIB
rflc/re1’
Carolina) of
Alabama).
Pennsylvania)
in
that can stably the appropriate
promote cell types
expression will need
be identified; viral enhancers and promoters found in many of the current vectors do not provide optimal expression in certain cell types and tissues. An example of using cellular transcription control regions within viral vectors to yield stable expression in the appropriate cell type is the demonstration by Ponnazhagan et al. [22]. These investigators showed that an a-globin promoter placed within the context of a recombinant AAV vector yielded more efficient expression in an erythroid cell line than either the HSV-TK or SV4O promoter. One possible approach to resolving the expression dilemma would be to “harness” the transcriptional activity of a known gene whose expression profile one would want the therapeutic gene to possess. One could harness the expression pattern of a known gene simply by homologously recombining a promoterless therapeutic gene downstream of the promoter of the known gene. Unfortunately, the homologous recombination techniques that are currently being applied to murine embryonic stem cells [23] are not sufficiently robust for use in human gene therapy protocols. However, advances in understanding the mechanism of homologous recombination may eventually put this approach into practice. A potential approach to utilizing cellular transcription control regions at their native locations would be gene delivery via enhancer-trap retroviruses. These retroviruses contam an enhancer deletion in the U3 region of the 3’ long terminal repeat (LTR). Retroviruses with such deletions self-inactivate transcription from the LTR promoter during transduction ofa target cell [24]. Enhancer-trap viruses have been shown to yield stable expression of a reporter gene in lymphoid cell types [25], whereas similar vectors utilizing the intact Moloney LTR have been shown to yield weak and unstable expression patterns [26]. In some cell types, expression from the Moloney LTR may even be suppressed [27].
Before gene therapy can become the strategy of choice in a wide variety of clinical settings, improvements in the efficiency of gene transfer into target cells and in the maintenance of expression from the relevant transferred gene must occur. The problem of efficient gene transfer will require not only further research to improve delivery systems and vector constructions but also a parallel effort to understand the biology ofthe target cells. Advances in understanding how target cells divide and differentiate may compensate for deficiencies in currently available delivery systems. For instance, if it could be determined how to cycle hematopietic
10
of
North
to
Prospects
Ic
Logan
Iowa)
cells.
promoter combinations of therapeutic genes
Many investigators have since attempted to identify similan mechanisms to interfere with the replication of HIV. Possible strategies for preventing HIV replication in infected cells include (1) trans-dominant negative HIV proteins (e.g., revMi0, gag), (2) ribozymes to genomic RNA, (3) antisense oligonucleotides to inhibit translation of HIV coding sequences, and (D) TAR decoys. In addition, some investigators have chosen immunotherapeutic approaches that are designed to augment the immune response to HIV and HIVinfected cells. For a more comprehensive review of gene therapy approaches for AIDS we refer the reader elsewhere [20, 21].
lhcrapcui
target
of Pennsylvania) of
liposornes.
wild-type HSV, suggesting that VP16 in the cells interfered with VPI6 and thus viral replication.
Future
the
(University (University
Peripheral
5.
Trials
of
Gene
lherapies
((Its
C’lL blood
AIDS
Mechanisiti
fibroblasts blood
for
lnvestigaior
Vaccine
1
cells
Trans
clones
Adoptive
1 cells
Inhibit
Intramuscular
dominant therapy
Gene
(USC)
Nabel
(University
Riddell
replication
Haubrich
therapy:
current
status
and
of
(University
Wong-Staal
Vaccine
Kerr and Mute
Galpin
Michigan) of
Washington)
(UCSD)
(UCSD)
future
prospects
213
Hence, self-inactivating retroviral vectors provide a means to trap and harness the transcriptional activity of cellular enhancers and thus could provide stable and sustained expression of a therapeutic gene. The one obvious drawback to this approach a cellular
is that enhancer
only
retroviral integrations will be transcriptionally
that active.
occur near However,
there is ample evidence that retroviruses prefer to integrate in transcriptionally active regions of the genome [28, 29], and thus the frequency of active integrations in target cells may not be substantially less with an enhancerless virus than with one with an intact enhancer region. In addition to improvements in delivery and expression technologies, future efforts will focus on new areas of gene therapy application. These include (1) identification and use of”new” resistance genes that will efficiently protect the bone marrow from alkylating agents and radiation; (2) intracellular immunization with therapeutic genes for use in adoptive immunotherapies against a variety of life-threatening infectious diseases (e.g., cytomegalovirus); and (3) novel adoptive immunotherapies employing T cells that have been gene modified to express “new” receptors (e.g., chimeric T cell receptors) [30].
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1994
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(1992)
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S.,
Nallari,
ML.,
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impedes
Nature 355, 452-454. E (1994) Progress towards 1, 13-26. for AIDS. Hum. Gene Ther. Snivasta,
A.
(1994)
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U.,
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E.E,
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(1986)
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