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GENE THERAPY
Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve
stable long-term correction of oxidase activity in peripheral blood neutrophils
Elizabeth M. Kang,1 Uimook Choi,1 Narda Theobald,1 Gilda Linton,1 Debra A. Long Priel,2 Doug Kuhns,2 and
Harry L. Malech1
1Laboratory
of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; and 2Neutrophil Monitoring
Laboratory, SAIC-Frederick, Frederick, MD
Chronic granulomatous disease (CGD) is
associated with significant morbidity and
mortality from infection. The first CGD
gene therapy trial resulted in only shortterm marking of 0.01% to 0.1% of neutrophils. A recent study, using busulfan conditioning and an SFFV retrovirus vector,
achieved more than 20% marking in
2 patients with X-linked CGD. However,
oxidase correction per marked neutrophil
was less than normal and not sustained.
Despite this, patients clearly benefited in
that severe infections resolved. As such,
we initiated a gene therapy trial for X-CGD
to treat severe infections unresponsive to
conventional therapy. We treated 3 adult
patients using busulfan conditioning and
an MFGS retroviral vector encoding
gp91phox, achieving early marking of 26%,
5%, and 4% of neutrophils, respectively,
with sustained long-term marking of 1.1%
and 0.03% of neutrophils in 2 of the
patients. Gene-marked neutrophils have
sustained full correction of oxidase activ-
ity for 34 and 11 months, respectively, with
full or partial resolution of infection in those
2 patients. Gene marking is polyclonal with
no clonal dominance. We conclude that
busulfan conditioning together with an
MFGS vector is capable of achieving longterm correction of neutrophil oxidase function sufficient to provide benefit in management of severe infection. This study was
registered at www.clinicaltrials.gov as
#NCT00394316. (Blood. 2010;115:783-791)
Introduction
X-linked chronic granulomatous disease (X-CGD) results from mutations of the CYBB gene encoding gp91phox required by neutrophils to
produce microbicidal oxidants. Patients develop recurrent lifethreatening bacterial and fungal infections.1 Management involves
prophylactic antibiotics, interferon-␥, and aggressive diagnosis and
treatment of infection. Allogeneic transplantation (BMT) is curative, but
consensus is lacking regarding indications to use what is perceived to be
a high-risk modality for a nonfatal disorder. Further, finding a siblingmatched donor in the setting of a genetic disease is a limiting factor.
However, outcomes improve when patients are transplanted earlier,
before numerous infections and end-organ damage, and when infection
free at the time of transplantation.
Gene therapy may be capable of providing protection from or
treatment of infection without the risks of BMT.2 Several publications reported protection from infection challenge after gene
therapy in gp91 and p47phox-deficient mouse models.3-7 Most
studies used myeloablative conditioning to achieve the engraftment
levels required for phenotypic correction. Gene-marking studies
with myeloablative conditioning in nonhuman primates have
achieved levels of long-term engraftment at levels that theoretically
would cure patients with CGD.8,9 Full engraftment is unnecessary
as demonstrated by X-CGD female carriers who vary in their ratio
of oxidase normal versus deficient neutrophils resulting from the
stochastic nature of X chromosome inactivation.10 Carriers with
more than or equal to 10% oxidase normal neutrophils are
generally infection free. However, in the nonhuman primate model,
gene marking has been significantly lower with nonmyeloablative
conditioning,11,12 particularly where there is no selective advantage
conferred by the transgene. Still, an early gene therapy trial for
X-CGD in humans resulted in detectable marking even without any
conditioning. In that trial, gene-corrected neutrophils occurred at
0.01% to 0.1% of total neutrophils measured at various time points,
but none persisted more than a year despite multiple infusions of
modified CD34s.13
In disorders where selective advantage is conferred by the
corrective transgene, such as X-linked severe combined immunodeficiency (SCID), there has been greater success.14,15 However, even
in adenosine deaminase deficiency SCID, the use of low-dose
busulfan (4 mg/kg) was required to obtain clinical benefit.16 In
2002, Ott et al17 initiated a clinical trial for X-CGD using a spleen
focus forming virus (SFFV)–based vector and busulfan at 8 mg/kg
for conditioning. The initial marking was greater than 20% in the
2 patients, although oxidase correction per gene-marked neutrophil
was significantly less than normal. The level of marked neutrophils
rose because of oligoclonal outgrowth of transduced cells with
vector inserted in the EVI1-MDS1 proto-oncogene. Unfortunately,
after 2 years, both patients developed myelodysplastic syndromes,
with one requiring BMT and the other dying of sepsis. Notably,
both patients’ infection at the time of gene therapy resolved, despite
the use of busulfan.17 Based on their observation of early benefit of
gene therapy for infection control, we initiated a gene therapy trial
to treat X-CGD patients with a severe infection not responsive to
conventional management. We used 10 mg/kg of busulfan followed
by autologous CD34s transduced ex vivo with an amphotrophic
pseudotyped vector encoding gp91phox. We describe here results in
3 patients treated to date.
Submitted May 19, 2009; accepted October 31, 2009. Prepublished online as Blood
First Edition paper, December 1, 2009; DOI 10.1182/blood-2009-05-222760.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
BLOOD, 28 JANUARY 2010 䡠 VOLUME 115, NUMBER 4
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KANG et al
Cell transduction
1
2 3 4 5 6
-6
-5
-4
-3
-2
-1
0
1
2
Figure 1. Study design. Patients’ cells were collected
and stored on a separate protocol. Day 0 indicates the
day of cell infusion. Cells were thawed on day ⫺5, and
transduction was begun on day ⫺4. The busulfan was
infused on days ⫺3 and ⫺2. Patients 2 and 3 received
palifermin given on days ⫺6 to ⫺4 and ⫺1 to 1. Patient 3
also started rapamycin on day ⫺1 and continued it for
30 days after transplantation.
Palifermin (patients 2 and 3 only)
Days of GCSF administration
(minimum two weeks prior
to gene therapy)
apheresis
Cells thawed (collected previously and stored)
Busulfan infusion
Rapamycin begins (patient 3 only)
Cell infusion
Methods
Clinical protocol regulatory review
All procedures and treatments were conducted under National Institutes of
Health protocol 07-I-0017 (the current protocol) or under protocol 95-I0134 (closed to accrual, but results from this protocol are discussed in this
paper). Both protocols were approved by the National Institute of Allergy
and Infectious Diseases Institutional Review Board, the National Institutes
of Health Institutional Biosafety Committee, the Food and Drug Administration, and the members of the Recombinant DNAAdvisory Committee of the
National Institutes of Health Office of Biotechnology Activities. Patients
were eligible for protocol 07-I-0017 if they had an infection not responsive
to standard therapy, proven gp91phox-deficient CGD, and no eligible human
leukocyte antigen-matched sibling donor. Availability of a human leukocyte
antigen-matched unrelated donor was not determined given its more
experimental nature and high risk in CGD patients with an ongoing
infection. Informed consent was obtained from all patients in accordance
with the Declaration of Helsinki.
Vector production
The amphotropic pseudotyped murine Moloney leukemia retrovirusderived vector encoding gp91phox cDNA (MFGS-gp91phox, supplemental
Figure, available on the Blood website; see the Supplemental Materials link
at the top of the online article) was produced from a 293-cell producer line
under Good Manufacturing Process conditions. Three production lots of the
same vector were used, with the first 2 made by Magenta for an earlier gene
therapy trial and revalidated for titer and potency. The third lot (BiorelianceInvitrogen) was made to supplement the current trial.
Autologous CD34ⴙ cells
Patients were enrolled on a separate Institutional Review Board-approved
protocol (94-I-0073) for collection of CD34⫹ hematopoietic stem cells.
They underwent standard mobilization and apheresis using filgrastim
(Amgen) at 10 to 16 ␮g/kg daily for 5 to 6 days (Figure 1). On day 5 and/or
6, a 15- to 25-L apheresis collection was performed. CD34⫹ cells were
selected using either the Isolex 300i (Baxter Healthcare; distributed by
Miltenyi Biotec) or CliniMACS Cell Selection System (Miltenyi Biotec)
cryopreserved and held in the National Institutes of Health, Department of
Transfusion Medicine until needed.
Transduction
Transduction was performed in the Cell Processing Section of the National
Institutes of Health Department of Transfusion Medicine. CD34s were
cultured and transduced in X-Vivo 10 (BioWhittaker Media, Cambrex Bio
Science) serum-free medium supplemented with 1% human serum albumin
and growth factors: interleukin-3 (5 ng/mL), stem cell factor (50 ng/mL),
thrombopoietin (50 ng/mL), and Fms-like tyrosine kinase 3 ligand (50 ng/
mL; all R&D Systems) in RetroNectin (recombinant fibronectin fragment;
Takara Bio)–coated X-FOLD bags (Baxter Healthcare). For each transduction, cells were resuspended in 90% neat vector supernatant at various cell
concentrations to maintain a multiplicity of infection of approximately
2. Cells were transduced daily times 4 days for 6 hours/day beginning at
16 to 18 hours of culture, and at 96 hours washed and resuspended in human
albumin supplemented phosphate-buffered saline for infusion.
Study design and conditioning
The extent of each patient’s infection was assessed before busulfan
administration. Using a central venous line, busulfan (Busulfex; Otsuka
America Pharmaceutical) 5 mg/kg was infused over 2 hours daily for 2 days
and levels measured at 0, 30, 60, 180, 240, 360, and 480 minutes after first
infusion (Quest Diagnostics). Area under the curve (AUC) and t1/2 were
assessed by Scott Penzak of the National Institutes of Health Clinical
Center Pharmacy using analytical software. The cells were given a
minimum of 24 to 48 hours after the busulfan was completed (day 0),
which, based on the calculated AUC and t1/2, was sufficient to ensure full
clearance of drug. Patients 2 and 3 were treated with palifermin (recombinant keratinocyte growth factor; Biovitrum) 60 ␮g/kg per day, for 3 days
before and after the busulfan. Patient 3 also received rapamycin (Wyeth
Pharmaceuticals) beginning on day ⫺1, with a loading dose of 5 mg given
3 times a day and then dosed to maintain a level of 10 to 15 mg/dL.
PCR quantification of vector marking
Semiquantitative real-time polymerase chain reaction (PCR) primers were
designed to anneal to the 5⬘ and 3⬘ long terminal repeats (LTRs) of the
MFGS vector.18 Forward primer sequence: CGC AAC CCT GGG AGA
CGT CC; reverse primer: CGT CTC CTA CCA GAA CCA CAT ATC C;
FAM labeled probe: CCG TTT TTG TGG CCC GAC CTG A. PCR was
40 cycles, melting temperature 95°C 15 seconds, and annealing temperature
60°C 60 seconds (PE Biosystems Realtime PCR 9600).
Detection of gp91phox expression
Intracellular gp91phox expression was quantified by flow cytometry. Briefly,
200 ␮L whole blood was lysed (ACK buffer), washed with phosphatebuffered saline/bovine serum albumin in 10mM ethylenediaminetetraacetic
acid buffer, and permeabilized using a kit (BD Biosciences). Permeabilized
leukocytes were resuspended in Solution B along with anti-gp91phox
monoclonal antibody (antibody 7D5, a generous gift from Dr Michio
Nakamura, Nagasaki, Japan) for 30 minutes, washed, and exposed to rat
anti–mouse fluorescein isothiocyanate-conjugated antibody (BD Biosciences)
for 30 minutes, and then analyzed using a FACSCaliber (BD Biosciences).
DHR flow cytometry assay of phagocyte respiratory burst
Analysis for dihydrorhodamine (DHR) oxidation was performed and
analyzed on a FACSCalibur using forward and side scatter characteristics to
define neutrophil and monocyte gates.19
Superoxide measurement
A quantiative ferricytochrome C reduction assay was used to measure
superoxide production at 10 and 60 minutes after stimulation.19
LAM PCR analysis of vector insert sites
Linear amplified mediated (LAM) PCR was performed20 on samples from
patients on this trial as well as archived samples from the 1998 trial
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BLOOD, 28 JANUARY 2010 䡠 VOLUME 115, NUMBER 4
X-CGD GENE THERAPY CORRECTS NEUTROPHIL OXIDASE
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Table 1. Patient characteristics
Patient no.
1
Age, y
28
Indication for gene therapy
Staphylococcal liver abscess
Mutation
461delA; N154fsX160 (frameshift)
Infection history
Recurrent infections including
previous bacterial
Medical treatment before
and during gene therapy
Intravenous meropenem,
vancomycin, and linezolid
abscesses of the liver
2
28
Paecilomyces chest wall infection
4 nucleotide deletion that spans
splice site 1150_E9(⫹2)delAA/gt,
Recurrent pneumonias
requiring thoracotomies
deletion of Exon 9 in mRNA
Intravenous ambisome then
intravenous voriconazole,
switched to posaconazole
and 5 Fluorocytosine, then
caspofungin added
3
19
Aspergillus lung infection
Nonsense mutation: C217A; R73X
Prior Aspergillus lung
Intravenous ambisome, then
infection, Burkholderia and
treated with voriconazole
Bartonella infections, and
and micafungin
osteomyelitis
Three patients with confirmed X-CGD were enrolled in the study using genetically modified cells to treat an unresolving infection. Patients’ infections, specific genetic
mutation, and infection history as well as the medical therapy used concurrently are listed.
(95-I-0134) using a primer pair set designed for the MFGS vector.
Linear PCR product using 5⬘ biotinylated primer was magnetic selected
and purified product mixed with Klenow polymerase and random
hexamers. Products were enzymatically cut with TASI (Fermentas; and
PUVI in some cases to remove the internal control band) and then
ligated to a linker cassette using a Fast-link DNA ligation kit (Epicentre
Technologies). Product was denatured to remove single-stranded DNA
and beads. Finally, exponential PCR was performed using linker and 5⬘
end-specific primers (LCI and LTR-R1 and LCIII and LTR-R2) and
analyzed on Spreadex gels for visualization. Individual bands were
sequenced using primers LCIII and LTR IV and TA cloning. Shotgun
cloning was also performed for integration site analysis using pooled
LAM PCR products and the TOPO TA cloning kit (primer sequences,
supplemental Methods). Clonal tracking was performed by designing
primers specific for LAM-detected insertion sites. Samples from various
time points were then reanalyzed to assess for presence of the clone with
the specific insert using seminested PCR.
Results
Patient characteristics
Three adult males with X-CGD (total deficiency of gp91phox
protein) were enrolled in this trial (Table 1). In 1995, Malech et al
Figure 2. Results from patient 1 during his first gene
therapy trial in 1998. Patient 1 from our trial was
enrolled previously on a gene therapy trial that used the
same vector but did not include conditioning. The patients received 2 separate infusions of cells as marked by
the arrows and the number of cells given ⫻ 104. Marking
was detectable early and increased after each infusion
but eventually was undetectable after 1 year.
initiated a clinical gene therapy trial for patients with p47phoxdeficient CGD without using conditioning.19 This was followed in
1998 by a trial for X-CGD, also without conditioning, the results of
which were reported in only preliminary detail.13 There, 6 X-CGD
patients were treated with the same MFGS-gp91phox vector as used
in the current trial. Those patients were given multiple infusions of
genetically modified cells but without conditioning (except for
patient 6 where it was used in conjunction with allogeneic
transplantation). Patient 1 from the current trial was also the third
patient treated in that previous study, allowing comparison of gene
transfer in the same person, first without conditioning (Figure 2)
and then 7 years later with busulfan, using the same vector lot
(results presented in subsequent sections throughout the article). In
the earlier study, he, along with 3 others (4 of 6 patients in that
study), produced very low but detectable numbers of oxidasepositive neutrophils, which rose transiently in the circulation after
each infusion but did not persist longer than 1 year. However,
2 of 6 patients in that previous study had no detectable marking.
For the current study, patient 1 presented with fevers caused by
Staphylococcus aureus liver abscesses not amenable to surgical
resection because of their size, location, and his previous complications from 5 prior liver resections. He was enrolled and treated in
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A
Absolute Neutrophil Count (K/µ
µl)
B
Days After Transplantation
Days After Transplantation
Figure 3. Effect of busulfan on neutrophil and platelet counts. (A) The absolute neutrophil count for each patient is plotted beginning before transplant (baseline) to day 50
after transplantation. Cell infusion was day 0 and the busulfan was given days ⫺3 and ⫺2. (B) The platelet count for each patient is plotted beginning before the busulfan and
until all patients no longer required any transfusions, day 48.
Functional correction of oxidase activity in circulating
neutrophils and monocytes
November 2006. Patient 2 had a Paecilomyces fungus pneumonia
extending into the chest wall and rib with recurrent accumulations
of pus and spontaneous drainage despite 2 years of combination
antifungal treatment and surgical resections. Patient 3 had Aspergillus fungus lung infection extending to ribs and vertebrae persisting
and expanding despite multiagent antifungal therapy for more
than 1 year.
At 2 to 3 weeks after infusion, DHR analysis of peripheral blood
circulating neutrophils and monocytes from patients 1, 2, and
3 demonstrated that 26%, 5%, and 4%, respectively, had become
oxidase normal. Figure 4 shows the results for neutrophils in each
patient over time, but monocytes had similar percentage correction
at each time point. In patient 1, the percentage of oxidase normal
cells declined slowly, reaching a plateau of 1% at 7 months; was
still 1.1% at 2 years after gene therapy (Figure 5); and remains
stable at 1.1% at 34 months (not shown). After initially producing
5% oxidase normal neutrophils, patient 2 had no DHR-positive
neutrophils and no PCR detectable inserts at 4 weeks or longer.
Like patient 1, patient 3 has persistence of oxidase normal
neutrophils in the circulation, declining from a high of 4% to a
steady-state level of 0.03% (Figure 4) that has persisted at this level
now with 11 months of follow-up (not shown).
Effects of busulfan
All patients developed neutropenia and thrombocytopenia (Figure
3). Time to neutrophil nadir varied from day 11 to 14, but all
patients had more than or equal to 500 000/␮L by day 28. Patient 3
required granulocyte colony-stimulating factor for prolonged neutropenia. All patients required platelet transfusions, with patient 3
requiring them to day 48. Patient 1 developed moderate mucositis.
With the addition of palifermin, patients 2 and 3 had little
mucositis. Overall, the patients tolerated the busulfan well.
Patient 2 received intravenous nutrition supplementation because of inanition from infection. All patients were maintained on
their infection-specific antimicrobials. The protocol was amended
to include rapamycin treatment for patient 3 because of early loss
of marking in patient 2. This was tapered off after 4 weeks, and
there was no evidence of toxicity from rapamycin.
Superoxide production
Before gene therapy, DHR assays of oxidase function in phorbol
myristate acetate (PMA)–stimulated neutrophils from all 3 patients
were uniformly negative with low mean fluorescence intensity
(MFI) similar to unstimulated cells. After gene therapy, at all time
points at which marking was detectable, the MFI of all 3 patients’
PMA stimulated neutrophils was not merely increased, but at each
assay was identical to the MFI of the PMA stimulated normal
neutrophils from a healthy volunteer (example of this comparison
shown in Figure 5). The ferricytochrome C reduction assay was
performed on patient neutrophils to provide precise quantitative
Graft characteristics
The characteristics of the autologous mobilized transduced CD34⫹
cell graft and busulfan AUC are shown in Table 2. Note that
significantly fewer cells were available for transduction for patient
3 compared with patients 1 and 2, as he mobilized poorly.
Table 2. Graft description
Patient no.
CD34/kg dose at start
CD34/kg dose after
transduction
CD34,
percentage
GP91,
percentage
Vector copy
number
Vector lot
number
Busulfan AUC,
␮M/min
11 324
1
14
43
85
73
4.05
1
2
13.7
71
⬎ 90
41
.8
2
7638
3
4.1
70
25
.005
3
7560
18.9
The CD34 doses as well as the marking of the graft as measured by either flow or GP91 expression or PCR for copy number, along with the busulfan levels for each patient
are given.
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BLOOD, 28 JANUARY 2010 䡠 VOLUME 115, NUMBER 4
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Figure 4. Percentage of DHRⴙ cells after cell infusion (day 0). Flow cytometric analysis was performed at various time points for each patient to assess for oxidase-positive
cells in the peripheral blood. Each patient had undetectable DHR before beginning the therapy. The percentage is plotted on a logarithmic scale with each patient having a
different range starting at 0.1 for patient 1, 0.001 for patient 2, and 0.01 for patient 3.
measurement of superoxide production. These assays confirmed
that the amount of superoxide produced by patient gene-corrected
neutrophils was the same as that from healthy volunteer neutrophils, when the data are corrected for the number of DHR-positive
neutrophils detected in the same sample (data not shown). This
demonstrates that the MFGS-gp91phox vector achieves sufficient
transgene protein expression in the cell to fully reconstitute
phagocyte oxidase activity.
Vector copy number and clonal tracking
PCR analysis of patient 1 total neutrophils (CD15 cells) at the
earliest time point, when 26% of neutrophils were DHR-positive,
showed a vector insert copy number of 1.335 per transduced cell.
The overall marking has declined to and stabilized at approximately 0.7%, consistent with the decline in the percentage of
DHR-positive cells. Marking was also detectable in the B-cell
fraction of patient 1 (0.6% currently). As expected, peripheral
T-cell marking in patient 1 was lower, starting at 0.057 and now
persisting at 0.002%. In addition, clonal tracking in this patient
shown in Figure 6 demonstrates the presence of the same clones at
both early (6 months) and late (2 years) time points in different
lineages, suggesting transduction and engraftment of long-term
repopulating progenitors (Figure 4). Patient 2 had early loss of any
Figure 5. Flow cytometry panel showing DHR analysis for
patient 1 at 2 years after gene therapy. Patient 1 who had the
best results continues to have approximately 1% DHR-positive
cells in the peripheral blood. The MFI for this patient’s cells is
consistently in the same range as the normal control run concurrently, indicating a close to normal level of oxidase production on a
per cell basis.
marking detectable by PCR, correlating with the DHR analysis.
Although PCR detectable marking in patient 3 has persisted,
correlating with a low level persistence of DHR-positive cells, the
very low marking precludes accurate PCR quantification or clonal
tracking.
Infection course
As shown in Figure 7, patient 1 demonstrated resolution of his liver
abscesses after gene therapy. Near the end of his prolonged course
of antibiotic therapy for liver abscess (8 months after gene
therapy), the patient developed an infection of the central venous
catheter that resolved quickly after removal of the catheter plus a
short course of antibiotics. For the next 2 years (now 34 months
after gene therapy), this patient has been free of any infection and
remains well on standard CGD antimicrobial prophylaxis.
Patient 2, who had early gene marking that quickly disappeared,
died of his invasive Paecilomyces fungus infection, dying almost
6 months after his gene therapy despite continuous intensive
antifungal therapy and allogeneic irradiated granulocyte transfusions. A donor search was initiated, but the best possible matches
were only 5 of 6 cord blood products. Unfortunately, the patient’s
infection continued to worsen, precluding even the possibility of a
double-cord transplantation.
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M
1
2
3
4
5
6
7
8
9
10
11
12
M
1
2
3
4
5
6
7
8
9
10
11
12
Clone #1
Inserted at Chr.1
Clone #3
Inserted at Chr. 11
M : DNA size marker
1 : CD3+ cells at 6 months after transplantation
2 : CD19+ cells at 6 months after
transplantation
3 : CD14+ cells at 6 months after
transplantation
4 : CD15+ cells at 6 months after
transplantation
5 : CD3+ cells at 1.5 year after transplantation
6 : CD19+ cells at 1.5 year after transplantation
7 : CD14+ cells at 1.5 year after transplantation
8 : CD15+ cells at 1.5 year after transplantation
9 :
10 :
11 :
12 :
CD3+ cells at 2 years after transplantation
CD19+ cells at 2 years after transplantation
CD14+ cells at 2 years after transplantation
CD15+ cells at 2 years after transplantation
Figure 6. Patient 1 clonal tracking PCR. Primers were designed based on inserts identified from the LAM PCR from patient 1, and a seminested PCR was performed on
samples obtained at various time points to determine their presence in the various lineages.
Patient 3 demonstrated significant radiologic evidence of regression of his Aspergillus infection followed by stabilization at
6 months after gene therapy with residual scarring and chronic
pulmonary changes. Given the extent of disease and the development of neutropenic fevers, this patient was treated with allogeneic
irradiated granulocyte infusions until recovery of an absolute
neutrophil count more than 500 cells/␮L, complicating interpretation of the role of gene therapy in resolution of his fungus infection.
Patients were not treated with granulocytes before the gene
therapy to avoid the development of alloimmunization. However,
as in both patients 2 and 3, they were used once gene therapy had
been performed. For patient 3, they were given for only the first few
weeks, a time that overlapped in part with the period of rapamycin
administration, and alloimmunization did not occur.
Insertional mutagenesis monitoring
All patients were closely monitored for evidence of clonality of
gene-marked cells. At no point has there been any evidence of
oligoclonality or clonal dominance. In particular, for patient 1 who
is now almost 3 years from treatment, the marking levels have
remained steady and monitoring by LAM PCR shows a diverse
polyclonal population with multiple bands seen in all marked
lineages. A bone marrow biopsy performed at 1 year and repeated
at approximately 3 years after gene therapy shows normal marrow
histology and karyotype. Of note, we have also performed an
analysis on archived samples from our 1998 X-CGD gene therapy
trial. Despite the very low marking, we cloned and characterized
42 unique sites from the first trial, and none involved EV11/MDS,
PRDM16 or Set BP1 nor were any sites found within 100 kb of
those genes. In addition, none of the original participants has developed
any evidence of MDS with follow-up more than 10 years.
Discussion
Image 1 pre gene therapy
Image 1 post gene therapy
Image 2 pre gene therapy
Image 2 post gene therapy
Indicates location of abscesses
Figure 7. Clinical results for patient 1. Shown here are the CT scans obtained
before gene therapy and 6 months after gene therapy for patient 1 who had
biopsy-proven Staphylococcal aureus liver abscesses as indicated by the arrow.
These disappeared, leaving only some scarring and regenerating liver as shown in
the corresponding post films.
Gene therapy has achieved lasting clinical benefit and restoration
of immunity for some primary immune deficiencies.14,16 However,
in disorders such as CGD where there is no selective advantage
provided by the corrective therapeutic gene, conditioning appears
necessary for long-term persistence of marked cells. In our 1998
gene transfer trial for X-CGD without conditioning, 4 of the
6 patients achieved only very low levels of gene marking, and that
marking did not persist. Busulfan conditioning in this current
protocol greatly improved initial marking and resulted in persistence of marking in 2 of the 3 patients. Patient 1 had early marking
peaking at 26% and has continued stable production of oxidase
normal cells composing more than or equal to 1% of circulating
neutrophils and monocytes from 7 months to almost 3 years at last
follow-up. Because this patient was also treated on the previous
gene therapy study without conditioning but using the same vector
lot, it is noteworthy that marking in the same patient in the previous
study never exceeded 0.055% and did not have any evidence of
genetically marked cells 9 months after the last infusion. Although
there are some differences in the transduction methods used in
these 2 studies, the initial presence of marked cells during the first
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BLOOD, 28 JANUARY 2010 䡠 VOLUME 115, NUMBER 4
trial with their subsequent disappearance versus continued presence of marked cells after busulfan supports the need for bone
marrow suppression to promote long-term engraftment.
Clinical benefit from adenosine deaminase (ADA)–SCID gene
therapy required some busulfan conditioning (4 mg/kg) despite
selective growth advantage conferred on lymphocytes. In that
setting, marrow engraftment of gene-marked CD34⫹ cell compartment was 5.1%, versus the 88% seen for the peripheral blood
T cells.21,22 Because no selective growth advance was anticipated
for correction of X-CGD, we used a higher but still not ablative
dose of busulfan (10 mg/kg). The source of CD34s, that is, bone
marrow versus mobilized peripheral blood, may also play a role;
however, cell dose, which is also a factor, is usually larger with
peripheral blood grafts. Ott et al chose busulfan at 8 mg/kg for their
trial as they also assumed a greater degree of conditioning was
necessary for long-term engraftment in the CGD setting.17 We
chose 10 mg/kg based on our own experience of using this dose in
an allogeneic transplantation for CGD. The difference between
8 and 10 mg/kg is unknown, although the increased effect may be
more than linear.22,23 Patient 1, who had the highest AUC perhaps
because of altered metabolism from his liver abscesses, had the
highest degree of marking. Despite the elevated AUC and his
underlying abscesses, he had no evidence of liver toxicity. Moreover, his duration of neutropenia, thrombocytopenia, and other side
effect profile were similar to the other 2 patients.
Our second patient had initial marking of 5% but lost that
marking extremely rapidly. Gene silencing occurs often in murine
gene transfer studies; it can also occur, although much less often in
human cells and human clinical trials24,25; and there was evidence
for silencing of transgene protein production in the Ott et al CGD
gene therapy trial.17 New vectors may incorporate transcriptional
insulators to reduce silencing.26,27 However, vector marking by
PCR analysis also became undetectable in patient 2 blood in
parallel with loss of oxidase activity, confirming that loss of graft
rather than gene silencing had occurred. We looked for development of an immune response as an etiology but did not find
evidence for this.
Nonetheless, development of an immune response to the
transgene, vector components, or other immunogens has clearly
been shown to occur in a large animal model of gene therapy.18
Thus, despite our inability to detect an immune cause of the rapid
loss of graft in patient 2, we could not ignore this possibility as we
planned the treatment of our third patient in this study. Because the
purpose of this gene therapy treatment trial is to provide salvage
therapy for an infection not controlled by standard therapy, there
was concern that we not interpret our failure to detect immunemediated graft loss as definitive evidence that immune elimination
of graft did not occur. We therefore sought to incorporate a way of
preventing immunity to the graft and enhancing tolerance without
significantly increasing risk to the patient. A consensus settled on
short-term treatment with rapamycin, a well-tolerated and widely
used immune suppressant with few side effects, a short half-life,
and a proven track record of inducing tolerance. For this reason,
patient 3 was treated with rapamycin starting 2 days before gene
therapy until 30 days after the gene therapy. The graft that patient
3 received had much fewer cells at lower transduction than patient 2, although initial marking of patient 3 was similar to that of
patient 2, 4.1% versus 5.0%, respectively. However, 0.03% of
circulating neutrophils and monocytes remain oxidase normal in
patient 3 at last assessment at 13 months. Although we cannot be
conclusive about any benefits from rapamycin, it was well tolerated
X-CGD GENE THERAPY CORRECTS NEUTROPHIL OXIDASE
789
and a persistence of corrected neutrophils was achieved that did not
occur in patient 2.
Regardless of the several mechanisms that may lead to cell
clearance, it is clear from our results that both the early and late in
vivo marking is directly affected by the ex vivo transduction
efficiency and total number of transduced cells infused. This effect
of transduction efficiency and cell dose has also been noted in the
NOD/SCID mouse xenograft model and in nonhuman primate gene
marking studies.28-30 Although profound effects on outcome may
also occur from vector insert effects such as observed in the study
using the SFFV vector, where gene marking of neutrophils was
very high in vivo despite only a 30% ex vivo transduction
efficiency, there were undesired side effects from this outgrowth.
We did not observe any evidence of insertional mutagenesis or
clonal expansion in our patients but do not think that vector
insertion driven expansion is a desirable way to enhance gene
marking. Although the degree of conditioning we achieved with
10 mg/kg of busulfan significantly improved both the short-term
and long-term marking, it is doubtful that higher doses will achieve
significantly higher levels of marking without increasing the
toxicity beyond acceptable levels. To achieve clinically beneficial
levels of marking, we think that maneuvers enhancing ex vivo gene
targeting of true hematopoietic stem cells, such as the use of
lentivirus vectors, may be one means of reaching this goal.31
The MFGS-gp91phox vector mediates sufficient production of
gp91phox transgene to fully correct oxidase function in an X-CGD
neutrophil. This was clearly demonstrated in our study by the DHR
and supporting quantitative assays of superoxide production. The
SFFV vector used in the Ott et al study17 mediated oxidase function
correction on a per neutrophil basis that was estimated by the
authors as only 15% to 30% of the amount produced by a normal
neutrophil. The level of oxidase production on a per neutrophil
basis may be as crucial for infection management as overall
transduction efficiency because microbial killing occurs within a
person cell’s phagosome. Although there are safety and other
advantages to lentiviral vectors that continue to drive an interest in
their development, our experience with production of gp91phox
proteins from lentivectors is that production levels to date have
been significantly much lower than what we can achieve with the
MFGS vector. Even if fewer cells are transduced, cells with normal
function can act synergistically with residing abnormal cells to
improve infection response.
Patient 1 had complete resolution of his massive liver infection.
This is a setting where experience has demonstrated32 that antibiotics alone, without the major surgery that was contraindicated in this
patient, would not have cured this infection. Although this does not
constitute proof that gene therapy cured his infection, a very strong
case can be made for significant clinical benefit to this patient from
the gene therapy. Furthermore, this patient has been infection free
for almost 23 months, the longest infection free period he has
experienced in the last 10 years despite being only partially
compliant with his maintenance antimicrobial prophylaxis.
Despite significantly less efficient gene correction than in
patient 1, patient 3 may also have obtained clinical benefit from the
gene therapy in that the progression of his Aspergillus infection that
was occurring before gene therapy stopped, significant resolution
of infection was documented, and the infection may be cured.
Patient 3 did receive a short course of allogeneic granulocyte
transfusions during the neutropenic period after busulfan conditioning, complicating any firm conclusion regarding the exclusive role
of the gene therapy in helping to control and resolve his fungus
infection.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
790
BLOOD, 28 JANUARY 2010 䡠 VOLUME 115, NUMBER 4
KANG et al
The 2 X-SCID trials33-36 and the Ott et al trial17 demonstrate that
retroviral vectors pose a risk of insertional mutagenesis. There does
appear to be a predisposition of the X-SCID phenotype to
oncogenesis with gene therapy and possibly a cooperation of the
common ␥ chain therapeutic gene itself to tumorigenesis.37,38 By
contrast, there has been no leukemia or myelodysplasia seen in any
ADA-SCID gene therapy patients. The CGD trial by Ott et al17
demonstrated a predisposition to outgrowth of clones with vector
insertions seen in EVI1, SetBP1, and PRDM16, but this may be in
part an effect of the strong myeloid enhancing element of the SFFV
vector LTR. Interestingly, these insertions were not seen in an
ADA-SCID patient treated with an SFFV-based vector.39 EVI1 has
been shown to be a target in other large animal studies using
non-SFFV gammaretrovirus vectors,40 although there was no
progression to clonal hematopoiesis or leukemia.41 Still, an ex vivo
mouse marrow myeloid immortalization study demonstrated that
SFFV-transduced clones had a higher rate of transformation,
compared with murine stem cell virus-transduced cells (C. Baum,
oral communication, June 2008), possibly explaining the difference
seen in our study compared with the CGD trial using the SFFV.17 In
our current study, we saw no evidence of any clonal outgrowth in
our 2 patients who we followed to date to 34 and 11 months of
follow-up. Analysis of more than 60 insertions characterized from
patient 1 to date did find 1 in EVI1 from 2 separate time points but
not in all analyses, nor does it predominate, and the overall pattern
of inserts remains polyclonal with no evidence of clonal outgrowth
and with continued evidence for normal hematopoiesis (supplemental Table).
A very recent brief report appearing in electronic print described
the outcome of gene therapy in an 8.5-year-old X-CGD child who
had severe Aspergillus pneumonia.42 The patient received busulfan
conditioning of 8.8 mg/kg, and CD34⫹ cells gene-marked ex vivo
at 31% to 34% (using the Ott et al vector17) were administered
intravenously and intraosseously. The level of early gene marking
of neutrophils reported at 26% to 29% was similar to that seen with
our patient 1, and as with him, their patient appeared to have
significant clinical benefit with control of his infection. Although
their report focused on neutrophil extracellular DNA nets as a
possible mechanism for benefit, less than 3 months of follow-up
from gene therapy is provided in the report, and level of oxidase
function per neutrophil cannot be determined from the paper.
However, their report supports the notion that gene therapy for
X-CGD can help control a life-threatening infection.
We show here the results of 3 patients treated with busulfan and
ex vivo genetically modified cells. Patient 1 had unequivocal
clinical benefit with continued production of 1% oxidase normal
cells in his peripheral blood now almost 3 years after treatment.
This represents the highest level of sustained marking with
production of functional gene product in neutrophils seen in any
patient to date beyond 1 year of follow-up with any disease that
does not have a selective advantage conferred by the corrective
gene or as a result of unintended clonal outgrowth. Patient 3 may
also have benefited in the control of his infection despite the
substantially lower level of long-term gene marking. All patients
tolerated the busulfan, which appears to be a critical element to
achieve engraftment of gene-corrected long-term repopulating
cells. Additional progress in the field will require higher rates of
gene transfer into pluripotent stem cells. Work to achieve this goal
has focused on new vectors, such as lentivectors,43 or novel
pseudotyping, such as RD114.43-45 Although gene therapy has not
yet cured patients with CGD, the field has made significant
progress with the demonstration that gene therapy can provide
clinical benefit to patients.
Acknowledgments
The authors thank the clinical staff who helped to care for the
patients as well as the Cell Processing Section of the Department of
Transfusion Medicine at National Institutes of Health for help with
the collection, processing, and transduction of the cell products.
Authorship
Contribution: E.M.K. designed and implemented the study and
wrote the paper; U.C. performed analyses, created figures, helped
with vector design and production, and reviewed the paper; N.T.,
G.L., D.A.L.P., and D.K. performed the majority of the assays in
the study; and H.L.M. helped design and support the study as well
as write the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Elizabeth M. Kang, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bldg 10-CRC, 6W Rm 6-3752,
9000 Rockville Pike, Bethesda, MD 20892; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2010 115: 783-791
doi:10.1182/blood-2009-05-222760 originally published
online December 1, 2009
Retrovirus gene therapy for X-linked chronic granulomatous disease
can achieve stable long-term correction of oxidase activity in peripheral
blood neutrophils
Elizabeth M. Kang, Uimook Choi, Narda Theobald, Gilda Linton, Debra A. Long Priel, Doug Kuhns
and Harry L. Malech
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