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Hemoglobin Gene Therapy for b-ThalassemiaArthur Bank, MD*
Human globin gene therapy is an exciting approach to curing homozygous b-thalassemia
(b-thalassemia, Cooley anemia) as well as sickle cell anemia. These diseases
are particularly suitable for this approach because the specific genetic defects that
cause them are known: sickle cell disease is caused by a point mutation in the human
b-globin gene; most b-thalassemia mutations are also caused by single nucleotide
changes, all of which lead to either decreased or absent normal b-globin protein.
Human b-globin gene therapy with autologous modified stem cells has been envisioned
for many years by patients, physicians, and scientists as a logical and ideal
way to cure the disease. However, it is only recently that some limited success has
been achieved.
The only cure for b-thalassemia (Cooley anemia) is allogeneic stem cell transplantation
(ASCT), using stem cells from adult peripheral blood, bone marrow, or umbilical
cord blood sources. ASCT is discussed in detail by Gaziev and Lucarelli elsewhere
in this issue , as well as by Kanathezhath and Walters. ASCT is limited by immunologic
differences between patients and potential donors; less than 30% of patients have
suitable donors. A curative result occurs in the most eligible patients who fit the criteria
for transplantation, most of whom are children. The potential development of graftversus-
host disease, a potentially life-threatening complication caused by immune
reactions, has tempered the use of ASCT, especially when a completely compatible
donor is not available.
There are 2 general approaches to providing normal b-globin function by gene
therapy in these disorders: correction of the DNA defect in the b-globin gene by
homologous recombination, or addition of a normal b-globin gene to the genome.
Gene correction has the great advantage of maintaining the b-globin gene in its native
This article is adapted from a chapter in Turning Blood Red: The Fight for Life in Cooley’s
Anemia, by Arthur Bank, published by World Scientific Publishing. The author is a founder,
equity holder, and consultant to Genetix Pharmaceuticals Inc, Cambridge, MA.
Columbia University, New York, NY, USA
* 4465 Douglas Avenue, New York, NY 10471.
E-mail address: ab13@columbia.edu
KEYWORDS
_ Thalassemia _ Cooley anemia _ Gene therapy _ Lentiviruses
_ Clinical trials
Hematol Oncol Clin N Am 24 (2010) 1187–1201
doi:10.1016/j.hoc.2010.08.002 hemonc.theclinics.com
0889-8588/10/$ – see front matter _ 2010 Elsevier Inc. All rights reserved.
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chromosomal environment, and thus is the preferred gene therapy approach.
However, homologous recombination occurs at too low a frequency at present to
be useful for human globin gene therapy.
Gene addition has been used successfully in human gene therapy clinical trials, with
viral vectors transferring and expressing corrective genes in human hematopoietic
cells. So-called g-retroviral vectors containing Moloney viral components have been
used to cure patients with severe immune disorders such as subacute combined
immunodeficiency (SCID) and adenosine deaminase deficiency.1–3 In these conditions,
the gene-corrected lymphocytes are naturally selected for survival and expansion
in preference to the patient’s own defective cells, and even low-level transduction
(infection) and expression of the corrective gene results in immune reconstitution of
the affected T lymphocyte compartment, and cure.
No such selection currently exists for gene-corrected hematopoietic stem cells
(HSC) containing and expressing the human b-globin gene in sickle cell disease or
b-thalassemia. Thus, high levels of normal b-globin transfer and expression are
required to cure these diseases.
GLOBIN GENE THERAPY
The current approach to human gene therapy for thalassemia is theoretically simple,
using autotransplantation. HSC are taken from the patient, a normal hemoglobin
(Hb) gene is added to the cells outside the body, and the human b-globin gene–corrected
cells are returned to the patient intravenously. They automatically home and
engraft in the marrow.
Gene therapy for b-thalassemia has been believed to be feasible since 1972, when
b-globin complementary DNA, (cDNA), a copy of globin messenger RNA, was
described.4,5 Then, it was believed that the globin cDNA itself could be used as the
source of the normal human b-globin gene sequences that could cure the disease.
However, in the 1980s, it became clear that, in addition to the coding sequences
present in globin cDNA, other important regulatory elements are required for successful
and high-level human b-globin gene expression. These sequences include the
intervening sequences within the gene, and regulatory sequences upstream and
downstream of the human b-globin gene. In the late 1980s, Grosveld and colleagues6
described important regulatory sequences far from the b-globin gene itself, called the
b locus control region (b LCR) that are necessary to provide high level of expression of
the human b-globin gene. The b LCR provides position-independent high-level
enhancement of globin expression, and its discovery was seminal in moving b-globin
gene therapy forward.6
Viruses as Vectors
Naked DNA can theoretically be used as the vector (or carrier) to transfer and express
genes in human gene therapy, including those for human b-globin. However, viruses
are much more efficient. Viruses are pieces of RNA or DNA wrapped in specialized
viral proteins: after infecting cells, viruses use the host cell’s molecular machinery to
encode specific viral proteins, and express and assemble the proteins into viruses.
Specific viral proteins on the surface of the viruses allow them to enter cells. After
infection and integration, the viral DNA directs the synthesis of more of viral proteins;
more viral particles assemble and are eventually extruded to infect more cells.
After infection, certain classes of viruses, adenoviruses, and, to some extent,
adeno-associated viruses, remain in the cytoplasm of cells; they do not enter the
nucleus and do not integrate into chromosomal DNA. These viruses are not useful
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for human blood stem cell gene therapy, a process that requires the corrective genes
to integrate into the patient’s chromosomes; this is necessary so that, when the genecorrected
stem cells divide, the corrective genes are transferred as part of the chromosomal
material and maintained in daughter stem cells.
RNA viruses, called retroviruses, are most useful for human gene therapy. These
viruses contain so-called gag, pol, and env gene sequences that lead to the production
of the proteins required by the virus: gag proteins produce the core proteins of the
virus; the pol gene specifies the enzyme, reverse transcriptase, which the viruses use
to make a DNA copy of their RNA; the env genes code for the proteins of the viral envelope.
These retroviruses also have genes that encode a protein called integrase, which
enhances the integration of viral genetic material into chromosomal DNA.
Intact replication-competent retroviruses produce more viral particles after integration,
often kill the cells they infect, release their viral particles from the cells, and infect
more cells. Replication-competent viruses are not desirable for human gene therapy;
not only can they kill the cells they transduce, but their integration at multiple sites in
host chromosomes can activate cellular oncogenes in a process known as insertional
mutagenesis. Instead, in human gene therapy, we use pieces of viruses, not intact
viruses, in such a way that they are incapable of generating intact viral copies of themselves,
while still carrying genes, such as the human b-globin gene, into cells and
integrating those genes into chromosomal DNA. We make so-called replicationincompetent
defective retroviruses (pseudoviruses) that, unlike their normal counterparts,
are unable to reproduce themselves after they have inserted their genetic
material into our chromosomes.
These defective viruses do not contain all of the proper genes and signals for new
wild-type viral production on a single piece of DNA or RNA, as in replication-competent
viruses. Defective viruses are created in so-called packaging cells; these are
tissue culture cells into which the genes that produce the necessary viral proteins,
gag, pol, env, and integrase, usually derived from Moloney leukemia viruses, are
added, The viral genes encoding these proteins are added to the packaging cells
on separate pieces of DNA called plasmids. The production of viral proteins in the
packaging cells leads to the formation of empty viral particles with no DNA or RNA
material capable of chromosomal integration.
When a suitable piece of DNA containing the corrective gene (in our case a human
b-globin gene–containing gene vector) is added to these packaging cells, so-called
producer cells are made. The nucleotide sequences on this vector plasmid are the
only ones that are integrated into the host chromosomes after viral integration. With
the production of gag, pol, env, and integrase proteins in the producer cells, retroviral
particles are formed containing the RNA encoding the corrective gene. The producer
cells then release intact viral particles into the medium. The pseudoviruses containing
the human b-globin gene transfer their globin gene sequences into target HSC, integrate
the gene sequences into chromosomal DNA, and allow the expression of potentially
curative human b-globin. To reiterate, these defective viruses, unlike their normal
counterparts, cannot reproduce themselves after they have inserted their genetic
material into our chromosomes; components of the material necessary to produce
intact viruses in this gene therapy system are on separate plasmids and cannot
generate normal infectious wild-type virus.
I believed many years ago that defective viruses containing the human b-globin
gene could be used as a pill that could be taken orally to cure sickle cell disease
and thalassemia. I believed that the pill would uncoat in the stomach; viral particles
would be released and enter the blood stream, exit at the right tissue location (in
this case, the bone marrow), and integrate into HSC DNA. Several tissue-specific
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viruses are known. Two examples are hepatitis virus, which contains envelope
proteins that target specific receptors on liver cells; and the acquired immune deficiency
syndrome (AIDS) virus, which only infects T lymphocytes. However, it has
not been possible to find viruses with envelopes that specifically target human HSC.
Instead, the approach has been to collect and concentrate HSC ex vivo, and use
envelope proteins that can enter many different cell types, including HSC. HSC are
obtained from either bone marrow or circulating blood, and our human b-globin
gene–containing retrovirus is added outside the body; this provides a great advantage
compared with other gene therapy applications in which the target organ cannot
be removed. Exposure of the virus is limited to the gene-targeted blood cells and there
is no danger of affecting non–blood cells. In addition, the ex vivo approach permits
high ratios of virus to relevant HSC, levels that might not be possible if the gene
therapy virus was given in vivo.
We used Moloney leukemia virus–based g-retroviruses, to transfer and express
human genes in 2 phase 1 human clinical trials to express a potential anticancer
gene, the multiple drug resistance (MDR) gene, in human HSC.7,8 For these trials,
we developed safe and efficient defective packaging cell lines to produce the defective
retroviruses.9,10 We achieved the expression of the MDR gene in the bone marrow
of patients, but at levels that were too low to be of clinical significance.7,8
Mouse Models
During the past 20 years, several groups have shown transfer and expression of
a human b-globin gene into mouse HSC, occasionally at high levels, with g-retroviral
vectors.11–14 In 1997, we reported one mouse that made 20% as much human
b-globin as mouse b-globin.14 However, this was a rare, significant, positive result
among many negative ones.
Trial and error has shown that efficient globin gene therapy is not reproducible using
g-retroviral vectors. This finding is primarily because the target cell, the HSC, is largely
quiescent; it divides infrequently. g-Retroviruses require cell division to move their
contents from the cytoplasm to the nucleus of cells. They are inefficient at having
the viral particles enter the nucleus of HSC and integrating into the chromosomes of
these cells that only occasionally divide.
To solve the problem of targeting nondividing human HSC, a special type of retrovirus,
a so-called lentivirus, a virus that does not require cell division to achieve HSC
target cell integration, is being used. Lentiviruses traverse the cytoplasm of nondividing
cells and, after reverse transcription, lentiviral particles can move from the cytoplasm
to the nucleus of cells without cell division. Sadelain and Leboulch pioneered
the use of lentiviruses in human globin gene therapy.15–18 Working independently,
they showed that the anemia in mice with diseases resembling human homozygous
b-thalassemia (Cooley anemia) and sickle cell disease can be alleviated significantly
using lentiviruses containing a normal human b-globin gene.15–18 These studies are
the impetus for the current human globin clinical trial in Paris and proposed trials.
SadelainandLeboulchwere responsible for anearlier criticalcontribution to the human
b-globin gene therapy field. They had previously shown that it was necessary to remove
certain specific nucleotide sequences from human b-globin IVS2 for the globin retroviral
vector to be appropriately reverse transcribed and expressed in target HSC.19,20
HUMAN GLOBIN GENE THERAPY
Human globin gene therapy is essentially autologous stem cell transplantation with
gene transfer. HSC from blood or marrow are removed from the patient,
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transduced with the corrective human b-globin gene–containing lentivirus, and
returned to the patient by vein (Fig. 1). This method overcomes the limitations
of allogeneic transplantation because there are no immunologic barriers to engraftment,
and, thus, many more patients are potentially eligible for treatment.
It is critical that human globin gene therapy specifically targets HSC, because these
are the only cells capable of both cell division and HSC maintenance, as well as of
erythroid differentiation. More differentiated transduced cells in the red blood cell
lineage will merely continue to differentiate for days, lose their nuclei and become
reticulocytes and mature red blood cells, and die.
In the past 2 decades, the environment for gene therapy research has been
more favorable in France than in the United States. The unexpected death of
a patient in a gene therapy trial at the University of Pennsylvania in 1999, using
another type of virus, an adenovirus, had made this area of research less
appealing to the scientific and medical community.21 The first largely successful
human clinical gene therapy trial was performed using g-retroviruses in Paris in
the late 1990s by Cavazzana-Calvo and colleagues.1,2 Nine of the 10 children
with X-linked SCID (X-SCID) in this trial were cured of the immunologic
deficiency.1,2
Fig. 1. Human Lentiglobin gene therapy. Bone marrow cells are harvested and purified by
Ficoll gradients and exposure to anti-CD34 antibodies. The CD341 cells are incubated
with growth factors (GF) including interleukin 3, thrombopoietin, Flt3 ligand and stem
cell factor for 24 to 48 hours, and then exposed to Lentiglobin, the human b-globin
gene–containing virus, for 24 hours. While the cells are being processed, the patient’s
own bone marrow is ablated using chemotherapy. Then the gene-altered cells are transfused
into the patient intravenously. (Adapted from Bank, A. Turning blood red: the fight
for life in Cooley’s anemia. Hackensack (NJ): World Scientific Publications; 2008. p. 216;
with permission.)
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Current Human Thalassemia Clinical Trial
The first and only ongoing human b-globin gene therapy clinical trial, is being performed
in Paris.22–24 A corrective human b-globin gene is being delivered using
a proprietary lentiviral product called Lentiglobin (Genetics Pharmaceuticals Inc.,
Cambridge, MA). (I am a cofounder of Genetix, the primary medical consultant to it
in the United States, and an equity shareholder). Lentiglobin contains the structural
human b-globin gene as well as its promoter and critical elements of the human
bLCR. Only these human b-globin gene–specific promoter-enhancer elements are
actively transcribed after Lentiglobin sequences are integrated in the genome of
gene-transduced target human HSC.22,24
The Lentiglobin gene–containing plasmid also contains the human immunodeficiency
virus (HIV) Rev response element and other DNA elements that enhance
nuclear migration of the viral DNA.23,24 The Lentiglobin plasmid is a so-called selfinactivating
(SIN) vector. It contains the 50 HIV long terminal repeat (LTR) with its
promoter and enhancer elements necessary for the transcription of Lentiglobin RNA
and for the reverse transcription of this RNA as a DNA copy. However, the 30 HIV
LTR promoter–enhancer sequences in the SIN vector are rendered inactive by deletions.
22–24 As in all retroviruses, when reverse transcription occurs, the 30 HIV LTR
becomes the 50 LTR sequences; in our case, the resulting Lentiglobin DNA 50 LTR
sequences are, thus, rendered inactive. This Lentiglobin SIN vector structure ensures
that no HIV promoter or enhancer sequences are active in transcription after integration
of Lentiglobin DNA into the host cell genome.
In addition, the Lentiglobin vector expresses so-called insulator sequences, short
DNA sequences placed on either side of the b-globin sequences in the Lentiglobin
plasmid, which prevent the activation of genes upstream or downstream of the insertion
sites of the Lentiglobin vector. The cells that produce Lentiglobin virus also
contain separate plasmids that encode and express lentiviral HIV gag and pol genes,
and the envelope protein, vesicular stomatitis viral protein (VSV-G). The Lentiglobin
virus used in the trial has been shown to express human b-globin at high levels in
target human HSC in mice.16,17
One disadvantage of currently used lentiviral production, including Lentiglobin in the
Paris trial, is that the expression of the viral HIV gag and pol proteins required are toxic
to the producer cells. This toxicity requires that the Lentiglobin vector plasmid is transfected
into a so-called transient packaging cell line system. In this system, the HIV
gag-pol, VSV-G env, and Lentiglobin plasmids are added to the packaging cells, 293T
monkey kidney cells, on separate plasmids, in small tissue culture dishes for 24 to 48
hours to transiently produce the Lentiglobin virus. Supernatants from several different
tissue culture dishes are harvested and constitute the infectious Lentiglobin virus used
in the trial. Pooled supernatants from the Lentiglobin virus–producing 293T cells are
further purified by chromatography before being used to transduce the HSC target cells.
The use of transient lentiviral systems is not as convenient as more desirable stable
producer lines developed with Moloney g-retroviral components, in which all of the
components of the virus are stably integrated into the packaging and producer cell
lines; these stable lines were used in the human MDR gene therapy trials.7,8 The
lack of toxicity of Moloney gag-pol expression permits single clones of g-retroviral
stable producer lines to be isolated and grown to large volumes without cell death
and used repeatedly. Several potentially useful stable lentiviral packaging lines have
been described recently in which the HIV gag and pol genes are stably integrated
into chromosomal DNA.25–29 In these lines, the toxicity of the HIV gag-pol genes is circumvented
by the transient and reversible expression of these genes using
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tetracycline control elements.25–29 However, none of these lines is currently used in
human clinical trials.
In the Paris gene therapy trial, CD341 cells are isolated from the bone marrow or
mobilized peripheral blood samples of patients; this CD341 cell purification eliminates
many differentiated cells, and concentrates the HSC. As previously noted, human
HSC are the only relevant population for human globin gene therapy because only
HSC can both self-renew and differentiate into erythroid cells, presumably for the
life of the patient. More differentiated nucleated erythroid cells differentiate and die
in days to weeks.
A Unique Human b-Globin Gene–containing Vector
In the ongoing phase 1 clinical trial in Paris, the human b-globin gene sequence in
Lentiglobin has been modified by mutating a single amino acid at position 87 of the
b-globin sequence.22,24 Hb containing b87 globin (Hbb87) has been shown to function
normally in its expression and oxygen-carrying capacity.16,17,30,31 It has also
been shown that Hbb87 acts like fetal Hb (HbF) in preferentially interfering with sickling
in studies of human sickled cells, and may be particularly useful in gene therapy trials
in patients with sickle cell disease.30,31
The major reason for using the b87 globin gene in Lentiglobin in patients with b-thalassemia
is that Hbb87 expression can easily be distinguished from that of normal Hb
(HbA). Because all patients with thalassemia continue to have significant amounts of
HbA in their blood as a result of transfusions after their transplantation, this distinction
is extremely useful.
The amount of new b87 globin gene in the patients’ cells can be measured by polymerase
chain reaction (PCR). New b87 globin protein is quantitated by high-pressure
liquid chromatography (HPLC). Thus, positive tests for the presence of the b87 gene
by PCR, and for b87 globin by HPLC, are clear measures of the success of human
b87 globin gene transfer and expression in the trial.
In the Paris trial, bone marrow is harvested from patients, CD341 cells isolated, and
Lentiglobin is transduced into these cells (see Fig. 1).22,24 If adequate Lentiglobin
transduction is documented in burst-forming unit, erythrocytes (BFU-E), and colonyforming
units-granulocyte macrophage (CFU-GM) cultures from the patient’s transduced
marrow samples, the patient then undergoes full myeloablation, and the
transduced cells are returned to the patient intravenously (see Fig. 1). The current principal
investigators (PIs) in the trial are Drs Leboulch and Marina Cavazzana-Calvo. Dr
Eliane Gluckman was an earlier co-PI.
Clinical trial protocols are a compromise between potential benefit and perceived or
known risk: the so-called benefit/risk ratio. In the Paris trial, we accepted the
increased risk of full marrow ablation to increase our chances for meaningful therapeutic
benefit. This risk was accepted because previous experience in human allogeneic
bone marrow transplantation (ABMT) has shown that less-than-complete ablation
of a recipient’s bone marrow is often insufficient to allow successful transplantation of
donor cells.32,33 It has been shown in mice that HSC transduced with retroviruses
compete unfavorably with wild-type HSC for marrow engraftment.34 This result
suggests that, in human gene therapy protocols using reduced-intensity marrow ablation,
the patient’s residual unmodified HSC will outcompete transduced gene–corrected
cells for marrow engraftment.
However, the risks of complete marrow ablation are also potentially greater than
using nonmyeloablative regimens, with longer periods of leukopenia and thrombocytopenia
after transplantation.
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Results to Date
Two patients with thalassemia have been treated to date on the current Paris
protocol.24 The first patient, TK, is a woman with severe thalassemia. After the gene
therapy procedure, she initially had evidence of a small amount of b87 globin gene
transfer by PCR that was transient and too low to be clinically significant. However,
her gene therapy treatment was not ideal because she received only one-third as
many gene-corrected cells as planned. She survived several weeks of very low white
blood cell and platelet counts, and was given antibiotics, white cell growth factors, and
platelet transfusions to ameliorate these complications. She also continued to receive
red blood cell transfusions to combat her anemia. She was eventually given an
untransduced backup bone marrow sample (collected and stored as part of the
protocol in case of the failure of the gene-corrected cells to engraft sufficiently). She
has now fully recovered her white cell and platelet function.
The second patient, PLB, is a 19-year-old man, doubly heterozygous for b-thalassemia
and HbE (a2bE
2).24 The bE gene acts like a b1 thalassemia allele and only
provides limited output of bE globin. The patient’s HbE gene output has always
been low (HbE<20% of normal HbA levels), and his limited HbE and HbF production
have not prevented him from severe lifelong anemia and its complications. He required
monthly transfusions since early childhood before the gene therapy procedure. He
also continued to need transfusions for several months after transplantation with the
b87-containing Lentiglobin vector.24 In contrast to patient TK, he had only a short
time after the gene therapy procedure of marrow hypoplasia during which his white
blood cell (WBC) count and platelet count were low.
Several months after transplantation, PLB began to significantly increase his
production of Hb b87.24 This increased production has continued in the last 19 months
(to June 2010), and he has not needed any blood transfusions during this time. He
is the first patient with a human Hb disorder to obtain clinical benefit and become
transfusion independent with human b-globin gene therapy.24 He currently has
approximately 9 to 10 g percent of Hb in his circulating blood, approximately one-third
being Hb b87, one-third human HbF, and one-third HbE.24 He has no residual transfused
HbA.
Safety Issues
During the past year, analysis of the clonal composition of patient PLBs reconstituting
b87-containing cells by linear amplification-mediated PCR has revealed that, although
reconstitution is polyclonal, a single clone is dominant. This clone has arisen from the
insertion of the Lentiglobin vector DNA into a specific gene, Hmga2; expression of Hb
b87 by this clone in erythroid cells is largely responsible for the Hb b87 production in
the patient.23,24 Approximately 10% of the patient’s CFU-GM and WBCs as well as
BFU-E contain the same insertion of Lentiglobin into Hmga2, indicating that the clone
is a multilineage clone, resembling a myeloid-biased HSC clone.24
Insertion of Lentiglobin into one of the introns of Hmga2 with a loss of intronic function
has occurred in this clone. This loss is similar to that occurring with other Hmga2
mutations, some of which are associated with clonal proliferation.24 Recent studies
have shown that clonality, with loss of Hmga2 introns, is more common than was
previously believed in patients in other human gene therapy trials35; in most of these
trials, these clones, presumably selected for their increased proliferative capacity, do
not continue to expand over time.35 The proliferation of the Lentiglobin-containing
HMGA2 clone in PLB has continued at a low level in the past year. It is unknown
whether the clone will expand, regress, or remain stable in the future.
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The goals of human globin gene therapy are safety as well as efficacy. Experience
from X-SCID trials is disconcerting in this regard. In these studies, although 9 of 10
patients in the Paris trial, and others in a similar trial in London, were cured of their
disease, several patients have subsequently developed leukemia as a result of insertional
mutagenesis.2 The unacceptable events in these trials are probably related, at
least in part, to the insertion of the curative gene in an HSC in the vicinity of an oncogene,
a gene whose expression can cause the uncontrolled growth of cells and
cancer.
In X-SCID, normal T lymphocytes lack a normal gC receptor gene, whose protein
product is necessary to fight certain types of infections.2 The curative gC receptor
gene used in these gene therapy trials is expressed from a g-retroviral promoterenhancer
LTR. In patients who developed leukemia, a rare cell inserted the gC
receptor gene and its powerful nonspecific promoter-enhancer near a known oncogene,
Lmo2, and activation of Lmo2 likely caused the emergence of the proliferative
leukemic clone.1
The design of the Lentiglobin vector used in the Paris study theoretically avoids the
potential problems of the gC vector in the X-SCID trial. As mentioned previously, first
and most importantly, viral enhancer elements are not activated in the SIN Lentiglobin
vector, as with the gC vector. Instead, only human b-globin gene–specific promoters
and enhancers direct human b-globin gene expression, and these are only active in
red blood cells. In addition, DNA sequences called insulators have been added to
the Lentiglobin vector to prevent any potential activation of oncogene sequences,
and the insertional mutagenesis seen in the X-SCID trial.
In patient PLB, the activation of HMGA2-mediated proliferation of a clone of HSClike
cells was not caused by activation of oncogenes by retroviral elements. The proliferative
clone is most likely the effect of the insertion of the Lentiglobin vector directly
into the intron of Hmga2 in one of the marrow stem cells during the initial transduction
of the patient’s HSC. Subsequently, the mutated clone was presumably selected for
proliferation; increased HMGA2 protein expression by the clone in PLB’s cells has
been confirmed in laboratory studies.24
Other Lentiglobin Human Gene Therapy Trials
Additional patients with severe thalassemia are currently being recruited to the Paris
trial. In addition, Dr Sadelain and his associates have planned a clinical trial with autotransplantation
to begin in the United States in the near future.18,36 This trial will use
a lentiviral human globin gene–containing vector designed by Dr Sadelain that is
somewhat different from Lentiglobin.
The only other human gene therapy trial using lentiviral vectors, other than the
human globin gene trial, has been reported recently in patients with the neurologic
disease, X-linked adrenoleukeukodystrophy (ALD).37 This is a rare disorder caused
by a genetic defect in the ABCD1 receptor gene in which there is deficiency of ALD
protein, an ATP-binding cassette transporter protein, required for normal myelination
of neurons. The disease is progressive and must be treated early in life to prevent
severe neurologic disability.37
ALD can be cured by ASCT. Macrophages produced by HSC have been shown to
migrate to the central nervous system (CNS), to become functional glial cells capable
of producing the normal required protein. In the gene therapy clinical trial of patients
with ALD, also performed in Paris, 3 boys with ALD deficiency were treated. CD341
cells were transduced with an ALD gene–containing lentiviral vector using procedures
similar to those used in the Paris human globin gene trial.37 In all 3 treated ALD
patients, there was a lack of progression of CNS demyelination compared with that
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in untreated patients with ALD.37 The results are encouraging and comparable with the
relative clinical benefit obtained using ASCT. To date, there has been no evidence of
specific clonal proliferation in any of the 3 ALD lentiviral vector–treated patients.
A New Homologous Recombination Approach to Thalassemia Gene Therapy
As discussed earlier, ideal human globin gene therapy for Cooley anemia as well as
sickle cell anemia would be gene correction: the correction of the single base mutation
in the DNA of the mutated human b-globin gene at its normal chromosomal position.
This correction can be accomplished by adding a vast excess of DNA containing the
normal b-globin DNA sequence to correct the mutant DNA sequence by homologous
recombination in the HSC of patients with sickle cell disease and thalassemia. Gene
correction has a great advantage compared with gene addition in that there is no
possibility of insertional mutagenesis because the corrective piece of DNA used is
short, biologically inert, and contains no viral elements; the only change in the patient’s
chromosomes is that the mutant b-globin gene is corrected.
However, as mentioned earlier, the frequency of gene correction occurring using
HSC is currently too low to be clinically useful. This shortcoming is primarily because
human HSC are limited in number and cannot be grown to large amounts in culture:
they differentiate preferentially into later blood cell elements rather than dividing and
reproducing themselves in large enough numbers to be useful for gene therapy.
Homologous recombination with gene correction has been successfully used for
many years to correct several gene defects in mice using embryonic stem (ES) cells,
the multipotential cells that are capable of producing any tissue of the animal.38,39 This
is because, in contrast with HSC, ES cells can be grown to large numbers without
altering their biologic properties. These large numbers of cells are required for gene
correction because it is such a rare event, and many individual cells must be isolated
and analyzed before sufficient gene-corrected cells can be obtained.
However, in the past 5 years 2 extraordinary advances have occurred that increase
the possibility that a gene correction strategy will eventually be used for gene therapy
for thalassemia and sickle cell disease. First, Takahashi and Yamanaka40 and Takahashi
and colleagues41 in Japan showed that human as well as mouse ES cells can be
derived by manipulating skin cells from each of these sources. In these experiments,
the addition and expression of just 4 genes (Klf4, Oct4, Sox2, and c-Myc) rewire the
circuitry of the differentiated skin cells so that they acquire many of the characteristics
of true ES cells. These reprogrammed cells are called induced pluripotent stem (iPS)
cells. Before this discovery, human ES cells could only be obtained from living human
embryos; this has been considered unethical by religious groups because it involves
the destruction of embryos. The use of the patients’ own skin cells to obtain ES cells
greatly diminishes these concerns.
Second, it has been shown that mouse iPS cells derived from skin cells and manipulated
in tissue culture can be used to cure mice with the equivalent of human sickle cell
anemia.42 In these experiments, skin cells from these mice were converted to iPS by the
addition of the 4 special genes mentioned earlier; short inert pieces of normal human
b-globin sequence DNA, containing sequences that correct the sickle cell mutation,
were added to the skin-derived iPS of these sickle mice; iPS were grown to large
amounts and the rare iPS in which the sickle cell mutation was corrected were isolated;
the globin gene–corrected iPS were grown to large amounts and, after being treated in
cell culture with growth factors and chemicals, became HSC.42 These human b-globin
gene–corrected HSC, originating from skin cells of mice, were then used to reconstitute
the ablated bone marrow of the sickle mice and largely cured their sickle cell anemia, as
assessed by correction of anemia and production of normal HbA.42
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Using an iPS strategy, as with our Lentiglobin gene therapy, there are no immunologic
barriers to transplantation because the skin cells originally used are derived from
the patient. However, as previously noted, a great advantage of this skin to ES cell
approach compared with our Lentiglobin addition gene therapy approach is that it
avoids the possibility of insertional mutagenesis because no functional DNA is added
to the patient’s chromosomes.
One of the genes initially used to convert skin fibroblasts to iPS cells was c-myc,
a known oncogene; increased expression of its protein product, c-Myc, is associated
with cancer. More recently, several advances, including protocols without increased
c-myc, have been made to address the problem.43–49 Such safer methods of generating
iPS cells from skin cells, avoiding c-Myc expression, and using small molecules
and/or the required proteins themselves rather than retroviral vectors producing those
proteins, may eventually make the use of human iPS cells more feasible for human
b-globin gene therapy. It will also be necessary to develop efficient methods for the
conversion of human iPS cells to human HSC to accomplish this goal.
Other Approaches to Thalassemia Gene Therapy
Another approach to curing b-thalassemia is to increase human HbF (a2g2) production.
Studies leading to clinical trials using a human g-globin–containing vector,
instead of a human b-globin vector, have been proposed.50
Recent success in understanding g-globin gene regulation and human g- to
b-globin switching in late fetal life has also suggested a different approach to thalassemia
and sickle cell gene therapy.51–53 Earlier, it had been shown that the protein
Ikaros was an important regulator of human g- to b-globin switching from data in
mouse models.51,54 More recently, it has been found that the action of a single human
gene, BCL11A, is much more potent than that of Ikaros.52,53
Like Ikaros, BCL11A is expressed primarily in adult-type hematopoietic cells and
forms chromatin remodeling complexes in these cells that suppress g-globin production.
In mice containing human g- and b-globin transgenes, it has recently been shown
that deletion of BCL11A leads to continued high-level production of HbF.52 If an antisense
strategy can be found to prevent BCL11A action in adult HSC, then continued
high-level HbF production might cure b-thalassemia and sickle cell disease. Recent
oligonucleotide and antisense RNA strategies may be useful in inhibiting BCL11A
expression in human HSC, and provide the basis for such a new globin gene therapy
approach.55–57
Strategies for the selection and enrichment of HSC containing and expressing
a curative human b-globin gene have been of interest for the past 2 decades.58–61
In this scenario, a retroviral vector plasmid containing both a selectable gene and
a human b-globin gene is used. The selectable gene, most commonly the human
MDR or methylguanine transferase (MGMT) gene, is one that is normally expressed
at low levels in human HSC. Transfer and expression of retroviral vectors that permit
high-level expression of MDR or MGMT in HSC then allow the preferential survival of
transduced cells, after systemic administration of certain drugs that kill cells expressing
only low levels of these proteins. Using this strategy, mouse HSC containing the
MGMT and MDR vectors alone have been shown to be preferentially selected by
drug administration in intact animals.59,60
However, human clinical trials using the human MDR or MGMT genes alone for HSC
selection have been unsuccessful in significantly enriching for gene-expressing
cells.7,8 Most recently, a lentiviral vector containing both the human g-globin gene
and the MGMT gene has been shown to select for cells containing and expressing
this bicistronic vector in mice.61
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SUMMARY
ASCT is the only currently available curative option for thalassemia and sickle cell
disease. Human b-globin gene therapy with autotransplantation of transduced human
HSC is an exciting alternative approach to potential cure. One patient with thalassemia
has recently been reported to show clinical benefit after lentiviral human globin gene
therapy. He has not required blood transfusions for almost 2 years. Most of the
patient’s gene correction and new human b-globin gene expression is caused by
the expansion of a single clone in which the corrective transgene is inserted into an
Hmga2 gene.
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