To assess the safety and efficacy of nonmyeloablative allogeneic transplantation in patients with HIV infection, a clinical protocol was initiated in patients with refractory hematologic malignancies and concomitant HIV infection. The results from the first 2 patients are reported. The indications for transplantation were treatment-related acute myelogenous leukemia and primary refractory Hodgkin disease in patients 1 and 2, respectively. Only patient 1 received genetically modified cells. Both patients tolerated the procedure well with minimal toxicity, and complete remissions were achieved in both patients, but patient 2 died of relapsed Hodgkin disease 12 months after transplantation. Patient 1 continues in complete remission with undetectable HIV levels and rising CD4 counts, and with both the therapeutic and control gene transfer vectors remaining detectable at low levels more than 2 years after transplantation. These results suggest that nonmyeloablative allogeneic transplantation in the context of highly active antiretroviral therapy is feasible in patients with treatment-sensitive HIV infection.

Allogeneic bone marrow transplantation (BMT) is a well-established approach with curative potential for a number of hematologic malignancies1; however, patients with concomitant HIV infection have generally been excluded from allogeneic BMT trials. Indeed, early allogeneic BMT trials as a primary treatment for HIV infection uniformly failed to control disease progression.2-4 The demonstration by Kolb et al5 that donor lymphocytes alone exert a powerful antileukemic effect, however, challenged the notion that high-dose chemoradiotherapy is necessary. This graft-versus-leukemia effect is most dramatically demonstrated among relapsed allogeneic BMT recipients receiving transplants for chronic myeloid leukemia; in these patients, a simple infusion of donor lymphocytes produces a complete and durable remission in the majority of treated patients6-8 and has led to intense interest and clinical activity investigating the role of nonmyeloablative transplant regimens for both malignant and nonmalignant disorders.9 10 We developed a clinical research trial to evaluate the use of cyclophosphamide/fludarabine–based conditioning in HIV- infected patients with an underlying malignancy, in whom an HIV-negative, HLA-matched sibling donor was available. Using this strategy, we also examined the possibility of efficient gene transfer of an HIV resistance vector carrying a dominant-negative mutant Rev at the level of the self-renewing stem cell.

The protocol was approved by the Institutional Scientific Review Board and the Institutional Review Board of the National Heart, Lung, and Blood Institute, the Institutional Biosafety Committee, the Center for Biologics Evaluation of the Food and Drug Administration, and the Office of Biotechnology Activities, and patients gave written informed consent. Eligibility included HIV positivity; an HLA-matched, HIV-negative sibling donor; and a hematologic malignancy meeting the standard indications for allogeneic transplantation in HIV-negative subjects.11 No restrictions were made regarding viral load or highly active antiretroviral therapy (HAART) sensitivity. Donor granulocyte colony-stimulating factor–mobilized peripheral blood was collected by apheresis on days 5 and 6. The day 5 apheresis product was CD34 enriched by means of a Nexell 300i (Nexell Therapeutics, Irvine, CA) automated immunomagnetic selection system for the recipient of genetically modified cells. Target CD34+cells were split equally for transduction with either (1) GCsapSL3rd3, containing a dominant-mutant or transdominant Rev (TdRev) engineered to inhibit viral replication through inhibition of wild-type Rev, a key HIV regulatory protein,12,13 or (2) a control vector encoding human GP91phox14 using previously established methods.15 Products not subjected to genetic modification, including the day 6 apheresis product for both patients, were cryopreserved without manipulation.

Patients were conditioned with a nonmyeloablative regimen16consisting of 60 mg/kg intravenous cyclophosphamide (IV) per day on days −7 and −6, followed by 25 mg/m2 fludarabine IV per day over 30 minutes on days −5 through −1. Cyclosporin was begun on day −4. HAART was discontinued 1 week prior to conditioning and was resumed when the patient was able to tolerate intake by mouth. Patients received antibiotic prophylaxis including acyclovir, bactrim, and norfloxacin as well as prophylaxis for mycobacterium aviumcomplex if indicated. Cytomegalovirus (CMV) reactivation was monitored by antigenemia and was treated preemptively with ganciclovir until day 100.17 

In vitro transduction efficiency was estimated by polymerase chain reaction (PCR) of DNA isolated from individual clonogenic progenitors derived from the end of transduction for genetically modified cells as previously described.18 Lineage-specific assessment of donor chimerism was determined as previously described.19Lineage-specific engraftment by genetically modified cells was determined by real-time PCR (Perkin Elmer, Branchburg, NJ) as previously described20 with the use of vector-specific primers.

Two patients have been treated to date. Their characteristics as well as the characteristics of the allograft are given in Table1. Both patients tolerated the procedure well and resumed HAART on day 0; they were discharged on day 9 and 11, respectively. In both patients, 100% donor chimerism was achieved by day 96 in both myeloid and lymphoid lineages. Lymphoid engraftment preceded myeloid engraftment and was similar to HIV-negative allogeneic transplant recipients conditioned with the same regimen.19 Both patients developed CMV antigenemia and were treated preemptively without evidence of disease. Restaging by bone marrow examination in patient 1 as well as computerized tomography and gallium scanning in patient 2 showed no evidence of disease in either. Grade II “acute” skin graft-versus-host disease (GVHD) developed in both beyond day 100, necessitating treatment with oral prednisone and continued cyclosporin. The observation of a clinical remission in concert with the development of acute GVHD in a patient with chemo-refractory Hodgkin is supportive of the concept of a graft-versus-Hodgkin effect.21-23 Control of GVHD in patient 2 was associated with relapsed Hodgkin disease in the retroperitoneum on day 180. Biopsy-proven central nervous system toxoplasmosis also developed and responded to treatment. However, despite withdrawal of immunosuppression, radiation therapy, interferon, and eventual administration of 1 × 107 donor lymphocytes per kilogram, the patient succumbed to progressive disease and died 12 months after transplantation. Patient 1 is currently being treated with cyclosporine and low-dose prednisone for limited chronic GVHD and remains in complete remission more than 2 years after transplantation.

The effect of nonmyeloablative allogeneic transplantation on HIV status is depicted in Figure 1. HIV was undetectable in both patients during extended follow-up with the exception of a brief period in patient 2 during which HAART was interrupted owing to persistent nausea. An acute febrile illness of 2 weeks' duration, with headache, cerebrospinal fluid pleocytosis, and a negative evaluation for infectious causes coincided with a rise in the HIV burden to 106 copies per milliliter. This episode may have represented acute HIV infection of the allograft,24underscoring the importance of continued antiretroviral therapy following transplantation. Reinstitution of HAART was associated with resumed viral control. CD4 counts rose above pretransplantation levels in both patients after transplantation. In patient 1, the CD4 count has risen as high as 450 cells per microliter, and the pattern of recovery is similar to that seen in HIV-negative allogeneic transplant recipients who received the same conditioning regimen (unpublished observations, November 1999).

Fig. 1.

Effect of transplantation on peripheral blood CD4 count and viral load.

Data for patient 1 are represented by dashed lines and symbols, and for patient 2 by solid lines and symbols. The CD4 count in cells per microliter is represented by squares for both patients and is plotted along the left y-axis. The circles represent the viral load in copies per milliliter, which is plotted along the right y-axis. Both variables are plotted against the days after transplantation. The horizontal lines represent the days of HAART therapy, and the horizontal arrows represent the beginning of the immunosuppression taper. The vertical arrow shows the commencement of interferon, and the arrowhead, the day of donor lymphocyte infusion given to patient 2. Patient 2 died 12 months after transplantation. The CD4 count for both patients remained at or above what it was prior to transplantation. The viral load remained undetectable at the latest time point measured.

Fig. 1.

Effect of transplantation on peripheral blood CD4 count and viral load.

Data for patient 1 are represented by dashed lines and symbols, and for patient 2 by solid lines and symbols. The CD4 count in cells per microliter is represented by squares for both patients and is plotted along the left y-axis. The circles represent the viral load in copies per milliliter, which is plotted along the right y-axis. Both variables are plotted against the days after transplantation. The horizontal lines represent the days of HAART therapy, and the horizontal arrows represent the beginning of the immunosuppression taper. The vertical arrow shows the commencement of interferon, and the arrowhead, the day of donor lymphocyte infusion given to patient 2. Patient 2 died 12 months after transplantation. The CD4 count for both patients remained at or above what it was prior to transplantation. The viral load remained undetectable at the latest time point measured.

Close modal

Recent improvements in gene transfer methodologies targeting the hematopoietic stem cell population have resulted in increased levels of genetically modified cells circulating after transplantation in relevant large animals, with levels of 5% to 10% or greater now achievable.25-27 While an equally high gene-transfer efficiency to the progenitor population was obtained as estimated by PCR, with 80% for the TdRev aliquot and 90% for the GP91phox aliquot, in vivo levels of circulating genetically modified cells were lower than those obtained in large animals. Overall marking levels of 0.01% were seen (Figure2). The differences in marking from the TdRev and control vector–transduced fractions did not suggest a survival or proliferative advantage to cells carrying the therapeutic gene; however, the absence of detectable HIV for the duration of the study suggests the absence of selective pressure as one potential explanation. Further, a threshold level of engraftment may be necessary to achieve a therapeutic effect. Finally, an effect by the genetically modified cells cannot be ruled out, as the recipient of genetically modified cells continues to show detectable marking from the TdRev vector with no detectable HIV. The low-level contribution toward hematopoeisis by genetically modified cells may also reflect competition by the unmanipulated graft. In murine and nonhuman primate competitive repopulation experiments, ex vivo cultured cells compete poorly against fresh cells, and cells cultured for longer periods compete poorly against cells cultured for shorter periods.25,28 29 

Fig. 2.

Percentage of transduced cells measured in the peripheral blood.

The gene-marking levels in patient 1 were analyzed by PCR. The percentage of cells positive for the GP91phox transgene is represented by dashed lines, and the percentage positive for TdRev, by solid lines. Marking in myeloid cells is shown as circles and in T cells as triangles. Overall marking averaged about 0.01% for both vectors and in both lineages.

Fig. 2.

Percentage of transduced cells measured in the peripheral blood.

The gene-marking levels in patient 1 were analyzed by PCR. The percentage of cells positive for the GP91phox transgene is represented by dashed lines, and the percentage positive for TdRev, by solid lines. Marking in myeloid cells is shown as circles and in T cells as triangles. Overall marking averaged about 0.01% for both vectors and in both lineages.

Close modal

A reluctance to consider allogeneic transplantation in HIV-infected patients may in part explain our slow accrual. However, these preliminary results suggest that allogeneic transplantation after nonmyeloablative conditioning represents a viable option for such patients. Further improvements in both gene transfer technology and allogeneic transplantation may allow application to patients with progressive AIDS without an underlying hematologic malignancy.

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 U.S.C. section 1734.

1
Armitage
JO
Bone marrow transplantation.
N Engl J Med.
330
1994
827
838
2
Angelucci
E
Lucarelli
G
Baronciani
D
et al
Bone marrow transplantation in an HIV positive thalassemic child following therapy with azidothymidine.
Haematologica.
75
1990
285
287
3
Giri
N
Vowels
MR
Ziegler
JB
Failure of allogeneic bone marrow transplantation to benefit HIV infection.
J Paediatr Child Health.
28
1992
331
333
4
Cooper
MH
Maraninchi
D
Gastaut
JA
Mannoni
P
Carcassonne
Y
HIV infection in autologous and allogeneic bone marrow transplant patients: a retrospective analysis of the Marseille bone marrow transplant population.
J Acquir Immune Defic Syndr.
6
1993
277
284
5
Kolb
HJ
Mittermuller
J
Clemm
C
et al
Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients.
Blood.
76
1990
2462
2465
6
Bar
BM
Schattenberg
A
Mensink
EJ
et al
Donor leukocyte infusions for chronic myeloid leukemia relapsed after allogeneic bone marrow transplantation.
J Clin Oncol.
11
1993
513
519
7
Drobyski
WR
Keever
CA
Roth
MS
et al
Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation: efficacy and toxicity of a defined T-cell dose.
Blood.
82
1993
2310
2318
8
Porter
DL
Roth
MS
McGarigle
C
Ferrara
JL
Antin
JH
Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia.
N Engl J Med.
330
1994
100
106
9
Giralt
S
Estey
E
Albitar
M
et al
Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: harnessing graft-versus-leukemia without myeloablative therapy.
Blood.
89
1997
4531
4536
10
Slavin
S
Nagler
A
Naparstek
E
et al
Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases.
Blood.
91
1998
756
763
11
Barrett
AJ
Mavroudis
D
Tisdale
J
et al
T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect.
Bone Marrow Transplant.
21
1998
543
551
12
Morgan
RA
Walker
R
Gene therapy for AIDS using retroviral mediated gene transfer to deliver HIV-1 antisense TAR and transdominant Rev protein genes to syngeneic lymphocytes in HIV-1 infected identical twins.
Hum Gene Ther.
7
1996
1281
1306
13
Mautino
MR
Keiser
N
Morgan
RA
Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by HIV-1-based lentivirus vectors expressing transdominant Rev.
J Virol.
75
2001
3590
3599
14
Malech
HL
Horwitz
ME
Linton
GF
et al
Extended production of oxidase normal neutrophils in X-linked chronic granulomatous disease (CGD) following gene therapy with gp(91phos) transduced CD34+ cells.
Blood.
92
1998
690a
15
Malech
HL
Use of serum-free medium with fibronectin fragment enhanced transduction in a system of gas permeable plastic containers to achieve high levels of retrovirus transduction at clinical scale.
Stem Cells.
18
2000
155
156
16
Childs
R
Clave
E
Contentin
N
et al
Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses.
Blood.
94
1999
3234
3241
17
Graber
C
de Almeider
KN
Childs
R
Barrett
AJ
Gill
VJ
Bennett
JE
CMV reactivation in nonmyeloablative HSCT.
Bone Marrow Transplant.
27
2001
775
18
Dunbar
CE
Cottler-Fox
M
O'Shaughnessy
JA
et al
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood.
85
1995
3048
3057
19
Childs
R
Chernoff
A
Contentin
N
et al
Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation.
N Engl J Med.
343
2000
750
758
20
Kang
EM
Hanazano
Y
Frare
P
et al
Persistent low-level engraftment of rhesus peripheral blood progenitor cells transduced with the fanconi anemia C gene after conditioning with low-dose irradiation.
Mol Ther.
3
2001
911
919
21
Dann
EJ
Daugherty
CK
Larson
RA
Allogeneic bone marrow transplantation for relapsed and refractory Hodgkin's disease and non-Hodgkin's lymphoma.
Bone Marrow Transplant.
20
1997
369
374
22
Carella
AM
Cavaliere
M
Lerma
E
et al
Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin's disease and non-Hodgkin's lymphoma.
J Clin Oncol.
18
2000
3918
3924
23
Anderlini
P
Giralt
S
Andersson
B
et al
Allogeneic stem cell transplantation with fludarabine-based, less intensive conditioning regimens as adoptive immunotherapy in advanced Hodgkin's disease.
Bone Marrow Transplant.
26
2000
615
620
24
Kahn
JO
Walker
BD
Acute human immunodeficiency virus type 1 infection.
N Engl J Med.
339
1998
33
39
25
Tisdale
JF
Hanazono
Y
Sellers
SE
et al
Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability.
Blood.
92
1998
1131
1141
26
Kiem
HP
Andrews
RG
Morris
J
et al
Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor.
Blood.
92
1998
1878
1886
27
Goerner
M
Bruno
B
McSweeney
PA
Buron
G
Storb
R
Kiem
HP
The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs.
Blood.
94
1999
2287
2292
28
Peters
SO
Kittler
EL
Ramshaw
HS
Quesenberry
PJ
Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts.
Exp Hematol.
23
1995
461
469
29
Qin
S
Ward
M
Raftopoulos
H
et al
Competitive repopulation of retrovirally transduced haemopoietic stem cells.
Br J Haematol.
107
1999
162
168

Author notes

John F. Tisdale, Bldg 10, Rm 9N116, 9000 Rockville Pike, National Institutes of Health, Bethesda, MD 20892; e-mail:johntis@intra.niddk.nih.gov.

Sign in via your Institution