Genetic modification of hematopoietic stem cells with genes that inhibit replication of human immunodeficiency virus-1 (HIV-1) could lead to development of T lymphocytes and monocytic cells resistant to HIV-1 infection after transplantation. We performed a clinical trial to evaluate the safety and feasibility of this procedure, using bone marrow from four HIV-1–infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34+ cells, performed in vitro transduction with a retroviral vector carrying arev-responsive element (RRE) decoy gene, and reinfused the cells into these subjects with no evidence of adverse effects. The levels of gene-containing leukocytes in peripheral blood samples in the 1 year after gene transfer/cell infusion have been extremely low. These observations support the potential of performing gene therapy for HIV-1 using hematopoietic cells, but emphasize the need for improved gene transfer techniques.

A POTENTIAL THERAPEUTIC approach to human immunodeficiency virus-1 (HIV-1) infection is the genetic modification of cells of a patient to make them resistant to HIV-1, termed intracellular immunization.1,2 Gene therapy for HIV-1 could target either mature peripheral blood T lymphocytes or hematopoietic stem cells. While T lymphocytes are more readily isolated and transduced, hematopoietic stem cells may be an attractive target because of their theoretical ability to generate a broad repertoire of mature T lymphocytes, as well as the monocytic cells (macrophages, dendritic cells, and microglia), which are also involved in HIV-1 pathogenesis. We have previously shown that overexpression of HIV-1rev-responsive element (RRE) sequences, as part of the transcript from the long-terminal repeat (LTR) of a retroviral vector, led to inhibition of HIV-1 replication in human T lymphocytes and in monocytic cells derived from transduced CD34+ hematopoietic progenitor cells. There was no evidence that expression of the RRE decoy adversely affected cell function.3,4 

STUDY DESIGN

We have performed a pilot clinical trial to assess the safety, feasibility, and efficacy of retroviral-mediated transfer of the RRE decoy gene into CD34+ cells isolated from the bone marrow of HIV-1–infected pediatric subjects. The protocol and informed consent document were reviewed by the Committee on Clinical Investigations (CCI) and the Institutional Biosafety Committee at Childrens Hospital Los Angeles, the Recombinant DNA Advisory Committee of the National Institutes of Health, and the Center for Biologics Evaluation and Review of the Food and Drug Administration.

Subjects were recruited from the Pediatric AIDS Program at Childrens Hospital Los Angeles. No specific criteria for CD4+ cell count or HIV-1 viral loads were used, to allow enrollment of subjects with a range of disease severity. Informed consent was obtained from parents and subjects 12 years or older or assent from subjects 7 to 11 years of age, following the guidelines of the CCI. Screening studies were performed to exclude subjects with severe organ dysfunction or contraindications to general anesthesia for bone marrow harvest. Bone marrow aspirates (5 to 10 mL) were obtained to determine the frequency of CD34+ cells and to grow autologous bone marrow stromal cells for support of the CD34+ cells during transduction. All subjects met the inclusion criteria of ≥1% CD34+cells in the marrow aspirate. The anti-retroviral drug regimens that the subjects received were those previously prescribed by their primary physicians and were not influenced by participation in the study.

RESULTS AND DISCUSSION

Four HIV-1+ pediatric subjects were enrolled and underwent the gene transfer protocol. Their age, weight, absolute CD4+ T-lymphocyte count, and plasma HIV-1 viral load are shown in Table 1. Subjects no. 1, 2, and 4 were teenagers who had been HIV-1+ for 12 to 16 years, after infection via transfusion. Subject no. 3 was an 8-year-old who was infected perinatally. Two of the subjects had very low CD4+ cell counts (2 and 6 CD4/μL) and two had CD4+ cell counts between 250 and 450/μL. All had high viral loads, despite two or three anti-retroviral drug therapy.

Table 1.

Patients’ Characteristics and Cell Yields

Patient No. Age (yr)Weight (kg) 
No.CD4μL
Plasma HIV (copies/mL) Bone MarrowPost-Isolex Posttransduction
BM vol (mL) 
Volumekg
%CD34+* 
No.CD34+(×106)kg
%CD34+ 
No.CD34+(×106)kg
 
No.cells(×106)kg
% Transduced CFU-C (G418-R/PCR+)
1  14 42  259  24,503  500  12.0  6.5  3.2  70.5 1.0  1.0  12/25.5  
2  16  38  6  30,762 425  11.0  5.4  0.65  75.6  0.76  0.35  7/13 
3  8  32  428  15,585  430  13.3  4.8  1.5 83.7  1.5  0.75  26/21.5  
4  17  53  96,528  226  4.3  5.1  1.3  69.7  0.28  0.05 22/16 
Patient No. Age (yr)Weight (kg) 
No.CD4μL
Plasma HIV (copies/mL) Bone MarrowPost-Isolex Posttransduction
BM vol (mL) 
Volumekg
%CD34+* 
No.CD34+(×106)kg
%CD34+ 
No.CD34+(×106)kg
 
No.cells(×106)kg
% Transduced CFU-C (G418-R/PCR+)
1  14 42  259  24,503  500  12.0  6.5  3.2  70.5 1.0  1.0  12/25.5  
2  16  38  6  30,762 425  11.0  5.4  0.65  75.6  0.76  0.35  7/13 
3  8  32  428  15,585  430  13.3  4.8  1.5 83.7  1.5  0.75  26/21.5  
4  17  53  96,528  226  4.3  5.1  1.3  69.7  0.28  0.05 22/16 
*

Post-Ficoll.

Aliquots of the CD34+ cells after transduction were plated in a methylcellulose-based colony assay in quadruplicate plates, with and without G418.23 After 14 days of culture, the numbers of hematopoietic colonies (CFU-C) were determined under phase-contrast microscopy. Individual colonies were collected from the methylcellulose and assayed for the presence of vector provirus by DNA-PCR. The percentages of CFU-C that were G418-resistant were determined by dividing the total number of colonies grown in the presence of G418 by the total number of colonies grown in the absence of G418 × 100%. The percentage of PCR+ colonies was determined by dividing the number of PCR+ colonies by the total number of colonies evaluated. All colonies grown from each patient (from L-RRE-neo transduced, LN transduced) were summated.

Bone marrow was obtained under general anesthesia from the bilateral posterior iliac crests and collected into RPMI 1640 with 500 U/mL heparin. Subjects remained in hospital overnight on the General Clinical Research Center after marrow donation and were discharged to home the next day without complications. The bone marrow was processed by centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden), followed by CD34+ cell isolation using the Isolex 300i (Nexell Therapeutics Inc, Irvine, CA).

Table 1 shows the volumes of bone marrow collected and cell yields. The target volume of ≥10 mL/kg was reached in subjects no. 1 through 3, but not in subject no. 4. The unprocessed marrow samples contained between 1.4% and 3.0% CD34+ cells, reconfirming in each case that the subjects met the inclusion criteria for marrow CD34+ cell content of ≥1%. The cell products produced by the Isolex 300i contained between 69.7% and 83.7% CD34+cells. The two subjects with extremely low CD4+ cell counts (nos. 2 and 4) had lower cell yields than the two subjects with higher CD4+ cell counts, although the small number of subjects preclude any firm conclusions.

To determine whether T lymphocytes expressing the RRE decoy have improved survival, we used an internal comparative marking technique, as has been used by others.5,6 One half of each patient’s cells were transduced with the L-RRE-neo vector that contains a 41-bp portion of the HIV-1 RRE sequences, inserted immediately upstream of the neo gene.3 The other half of their cells were transduced with the control LN vector. Both vectors were packaged as clones in PA317 amphotropic packaging cells.7 Clinical grade supernatants were produced by the Gene Vector Laboratory at Baylor College of Medicine in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum. Titers of the supernatants were 1 to 3 × 106/mL based on G418-resistance of serial dilutions on 3T3 cells. Testing for adventitious agents and replication-competent retroviruses (RCR) were performed by the Gene Vector Laboratory at Baylor College of Medicine or by Microbiologic Associates (Rockville, MD) and were all negative.

The CD34+ cells were divided into two portions of equal cell numbers, one for transduction with L-RRE-neo and one for transduction with LN. Autologous bone marrow stromal cells (irradiated with 2,000 cGy, 1 day before use) were used as support layers during transduction of cells from subjects no. 1 through 3.8 For subject no. 4, recombinant fibronectin CH-296 (RetroNectin; Takara Shuzo Co, Ltd, Otsu, Shiga, Japan) was used instead of autologous stroma. CD34+ cells were transduced for three 24-hour transduction cycles with recombinant cytokines interleukin-3 (IL-3) (20 ng/mL; Novartis, Piscataway, NJ), IL-6 (50 ng/mL; Novartis), and Steel factor (100 ng/mL; R & D Associates, Minneapolis, MN) and protamine sulfate (4 μg/mL; Lyphomed, Deerfield, II), as described previously.4 Because subject no. 4 had a poor yield of marrow and CD34+ cells, no conclusions can be drawn about any differences in cell recovery or transduction using stroma versus fibronectin CH-296.

After the three transduction cycles, the nonadherent cells and culture medium were collected from the flasks, and then the adherent cells were collected from the flasks using enzyme-free cell dissociation buffer (GIBCO-BRL, Bethesda, MD). The nonadherent and adherent cells were combined and washed four times with Hanks’ balanced salt solution with 1 U/mL heparin and resuspended in 15 mL plasma-lyte A with 1 U/mL heparin. The two cells pools transduced separately with L-RRE-neo and LN were combined for a total final volume of 30 mL, which constituted the final cell product. Patients were monitored clinically during the cell infusion and afterward overnight.

All monitoring gram stains, bacterial, fungal, and mycoplasma cultures, endotoxin assays, and measurements for RCR in culture medium and cell pellets were negative. There was no detectable HIV-1 in the transduced CD34+ cell pellets or culture medium by DNA polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR, respectively.

Subjects received their cells by intravenous infusion without complications, based on clinical monitoring and laboratory studies. Postinfusion evaluations showed no perturbations in vital signs, complete blood count (CBC), or chemistry panels. There was no evidence that the patients were exposed to RCR, by assay for infectious RCR from their peripheral blood mononuclear cells (PBMC) (days +1, +7, and +30) or by Western blot analysis for the development of serum antibodies reacting against Moloney murine leukemia virus (MoMuLV) proteins (months +1, +2, +3). There were no detectable changes in any of the subject’s plasma HIV-1 levels from the marrow harvest or cell reinfusion. Thus, the subjects did not receive a significant inoculum of HIV-1 upon reinfusion of their transduced cells. Apparently, the transduced cells do not serve as a site of HIV-1 replication in vivo, despite their activation in vitro from culture with the recombinant cytokines used to stimulate stem cell proliferation to allow retroviral-mediated transduction.9 

Transduction of clonogenic progenitor cells within the CD34+ cells was evaluated by growing CFU-C colonies and measuring the percentage that were G418-resistant and the percentage that contained vector DNA by DNA-PCR of individual colonies which were not selected in G418 (Table 1). Transduction efficiencies were between 7% and 30% by either assay and the percentages of transduced colony-forming unit-cells (CFU-C) measured by either G418-resistance or PCR were not significantly different (P = .77). The two vectors (L-RRE-neo and LN) led to similar extents of transduction (data not shown, P = .87).

Venous blood samples were obtained at serial times after the infusion of transduced CD34+ cells to measure the percentage of PBMC containing vector sequences. Positive samples have been seen in all four subjects (Fig 1). Cells containing both the L-RRE-neo and LN vectors were detected on the day after cell infusion in two of the subjects, at frequencies of 0.03% and 0.01% (1 to 3 gene-containing cells per 10,000). With the exception of those day +1 samples, all subsequent samples were negative for the L-RRE-neo vector. Cells containing the LN vector were detected in some later samples for as long as 270 to 330 days, at frequencies of 1 cell per 100,000.

Fig. 1.

Gene frequency in PBMC after infusion of transduced CD34+ cells. Isolation of genomic DNA from patient leukocytes and PCR amplification and analysis were performed as described previously.11 To develop quantitative standards, mixtures of genomic DNA extracted from CEM cells containing one proviral copy of either the LN or the L-RRE-neo vectors were diluted in genomic DNA extracted from nontransduced cells. These standards plus multiple samples of DNA from nontransduced PBMC were analyzed with each assay of patient samples. A single pair of PCR primers was made to simultaneously detect the presence of sequences from the LN and L-RRE-neo vectors. The 5′ primer was complimentary to the MoMuLV Psi region (5′-CGAGACCTCATCACCCAGGTTAAG-3′ sense) and the 3′ primer was complimentary to the neo gene (5′-CATCAGAGCAGCCGATTGTCTG-3′ antisense). This pair of primers produces a product of 391 bp from the LN vector (N) and 451 bp from the L-RRE-neo vector (R); the increase in the L-RRE-neo product being caused by the presence of the RRE sequences immediately 5′ of the neo gene, within the span of the primer pair. An oligonucleotide (5′-TCGATCCTCCCTTTATCCAGCC-3′) complimentary to a sequence present in the resultant PCR products from each vector was labeled with -32P-dATP and used as probe.

Fig. 1.

Gene frequency in PBMC after infusion of transduced CD34+ cells. Isolation of genomic DNA from patient leukocytes and PCR amplification and analysis were performed as described previously.11 To develop quantitative standards, mixtures of genomic DNA extracted from CEM cells containing one proviral copy of either the LN or the L-RRE-neo vectors were diluted in genomic DNA extracted from nontransduced cells. These standards plus multiple samples of DNA from nontransduced PBMC were analyzed with each assay of patient samples. A single pair of PCR primers was made to simultaneously detect the presence of sequences from the LN and L-RRE-neo vectors. The 5′ primer was complimentary to the MoMuLV Psi region (5′-CGAGACCTCATCACCCAGGTTAAG-3′ sense) and the 3′ primer was complimentary to the neo gene (5′-CATCAGAGCAGCCGATTGTCTG-3′ antisense). This pair of primers produces a product of 391 bp from the LN vector (N) and 451 bp from the L-RRE-neo vector (R); the increase in the L-RRE-neo product being caused by the presence of the RRE sequences immediately 5′ of the neo gene, within the span of the primer pair. An oligonucleotide (5′-TCGATCCTCCCTTTATCCAGCC-3′) complimentary to a sequence present in the resultant PCR products from each vector was labeled with -32P-dATP and used as probe.

The failure to achieve significant transduction and engraftment of hematopoietic stem cells in this trial stands in contrast to our results using similar methods of gene transfer into CD34+cells from the umbilical cord blood of infants with adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID).10,11 In the ADA-deficient subjects, the continued presence of gene-containing cells of myeloid and lymphoid lineages for over 5 years showed that long-lived stem cells were transduced and engrafted. The difference may reflect the presence of a modest percentage of hematopoietic stem cells in cord blood which are actively undergoing cell replication, in contrast to the near-absence of replicating stem cells in bone marrow.12 The spontaneously dividing stem cells in umbilical cord blood would be susceptible to transduction by MoMuLV retroviral vector, whereas the quiescent stem cells from bone marrow would not. It is also possible that immunologic responses to either the neomycin phosphotransferase protein encoded by the vectors or to components of the cell culture medium may have led to in vivo elimination of the transduced cells.

Recent incremental improvements in retroviral-mediated gene transfer into human hematopoietic stem cells (HSC) have been achieved, using gibbon ape leukemia virus (GALV) pseudotypes, “mobilized bone marrow,” recombinant fibronectin support, new cytokines (Flt-3 ligand, thrombopoietin), and manipulation of cell-cycle kinetics.13-18 Combinations of these techniques have resulted in modest, yet significant, increases in gene marking in primate stem cell transplant models (eg, 10%, up from the previous ceiling of 0.1% to 1.0%).19,20 However, even higher levels of gene transduction of stem cells are likely to be needed for applications to many genetic diseases and acquired immunodeficiency syndrome. The development of vectors based on lentiviruses, such as HIV-1 or feline immunodeficiency virus (FIV), holds the promise of producing increased transduction of HSC, due to their ability to transduce quiescent cells such as neurons, hepatocytes, and macrophages.21,22 The safety and feasibility shown by the present study will allow future studies to evaluate these newer methods for their potential efficacy.

ACKNOWLEDGMENT

We thank the patients and families who participated in this study. Essential reagents were generously provided by Katie Harding (Novartis, Piscataway, NJ), John MacManus and Virginia Mansour (Baxter/Nexell, Irvine, CA), and Dr Ikunoshin Kato and Setsuko Yoshimura (Takara, Otsu, Shiga, Japan).

These studies were performed in the General Clinical Research Center at Childrens Hospital Los Angeles (3 MO1 RR0043-35S1) with support by the GCRC Gene Therapy Core Laboratory, a SPIRAT grant from the National Institute of Allergy and Infectious Diseases (1U19 AI36606), and research grants from the Pediatric AIDS Foundation (no. 30001-17-GT) and the T.J. Martell Foundation. D.B.K. is the recipient of an Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

REFERENCES
1
Baltimore
D
Intracellular immunization.
Nature
335
1988
395
2
Gilboa
E
Smith
C
Gene therapy for infectious diseases: The AIDS model.
Trends Genet
10
1994
139
3
Bahner
I
Kearns
K
Hao
QL
Smogorzewska
M
Kohn
DB
Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits HIV-1 replication in the myelomonocytic cells produced in long-term culture.
J Virol
70
1996
4352
4
Bauer
G
Valdez
P
Kearns
K
Bahner
I
Wen
SF
Zaia
J
Kohn
DB
Inhibition of HIV-1 replication after transduction of G-CSF-mobilized CD34+ cells from HIV-1–infected donors using retroviral vectors containing anti–HIV-1 genes.
Blood
89
1997
2259
5
Woffendin
C
Ranga
U
Yang
Z-Y
Nabel
GJ
Expression of a protective gene prolongs survival of T cells in human immunodeficiency virus-infected patients.
Proc Natl Acad Sci USA
93
1996
2889
6
Morgan
RA
Walker
R
Carter
C
Bechtel
C
Bunnell
B
Fellowes
V
Muul
L
Leitman
S
Lane
C
Blaese
RM
Preferential survival of CD4 T-cells engineered with anti-HIV genes in patients enrolled in a gene therapy trial in HIV-1 discordant identical twins. American Society of Gene Therapy
1998
44a
WA
Seattle
(abstr)
7
Miller
AD
Buttimore
C
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol Cell Biol
6
1986
2895
8
Wells
S
Malik
P
Pensiero
M
Kohn
DB
Nolta
JA
The presence of an autologous marrow stromal cell layer increases glucocerebrosidase gene transduction of long term culture initiating cells (LTCIC) from the bone marrow of a patient with Gaucher disease.
Gene Ther
2
1995
512
9
Kearns
K
Bahner
I
Bauer
G
Wei
SF
Valdez
P
Wheeler
S
Woods
L
Miller
R
Casciato
D
Galpin
J
Church
J
Kohn
DB
Suitability of bone marrow from HIV-1–infected donors for retroviral-mediated gene transfer.
Human Gene Ther
8
1997
310
10
Kohn
DB
Weinberg
KI
Nolta
JA
Heiss
LN
Lenarsky
C
Crooks
GM
Hanley
ME
Annett
G
Brooks
JS
El-Khoureiy
A
Lawrence
K
Wells
S
Shaw
K
Moen
RC
Bastian
J
Williams-Herman
DE
Elder
M
Wara
D
Bowen
T
Hershfield
MS
Mullen
CA
Blaese
RM
Parkman
R
Engraftment of gene-modified cells from umbilical cord blood in neonates with adenosine deaminase deficiency.
Nat Med
1
1995
1017
11
Kohn
DB
Hershfield
MS
Carbonaro
D
Shigeoka
A
Brooks
J
Smogorzewska
EM
Barsky
LW
Chan
R
Burotto
F
Annett
G
Nolta
JA
Crooks
G
Kapoor
N
Elder
M
Wara
D
Bowen
T
Madsen
E
Snyder
FF
Bastian
J
Muul
L
Blaese
RM
Weinberg
K
Parkman
R
T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates.
Nat Med
4
1998
775
12
Hao
QL
Shah
AJ
Thiemann
FT
Smogorzewska
EM
Crooks
GM
A functional comparison of CD34+CD38− cells in cord blood and bone marrow.
Blood
86
1995
3745
13
Kiem
HP
Heyward
S
Winkler
A
Potter
J
Allen
JM
Miller
AD
Andrews
RG
Gene transfer into marrow repopulating cells: Comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons.
Blood
90
1997
4638
14
Dunbar
CE
Seidel
NE
Doren
S
Sellers
S
Cline
AP
Metzger
ME
Agricola
BA
Donahue
RE
Bodine
DM
Improved retroviral gene transfer into murine and rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.
Proc Natl Acad Sci USA
93
1996
11871
15
Hanenberg
H
Xiao
XL
Dilloo
D
Hashino
K
Kato
I
Williams
DA
Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.
Nat Med
2
1996
876
16
Shah
AJ
Smogorzewska
EM
Hannum
C
Crooks
GM
Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38− cells and maintains progenitor cells in vitro.
Blood
87
1996
3563
17
Dao
MA
Hannum
CH
Kohn
DB
Nolta
JA
Flt3 ligand preserves the ability of human CD34+progenitors to sustain long-term hematopoiesis in immune-deficient mice afer ex vivo retroviral-mediated transduction.
Blood
89
1997
446
18
Dao
MA
Taylor
N
Nolta
JA
Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells.
Proc Natl Acad Sci USA
95
1998
13006
19
Kiem
H-P
Andrews
RG
Morris
J
Peterson
L
Heyward
S
Allen
JM
Rasko
JEJ
Potter
J
Miller
AD
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
20
Tisdale
JF
Hanazono
Y
Sellers
SE
Agricola
BA
Metzger
ME
Donahue
RE
Dunbar
CE
Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability.
Blood
92
1998
1131
21
Naldini
L
Blomer
U
Gallay
P
Ory
D
Mulligan
R
Gage
FH
Verma
IM
Trono
D
In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.
Science
272
1996
263
22
Poeschla
E
Corbeau
P
Wong-Staal
F
Development of HIV vectors for anti-HIV gene therapy.
Proc Natl Acad Sci USA
93
1996
11395
23
Nolta
JA
Kohn
DB
Comparison of the effects of growth factors on retroviral vector-mediated gene transfer and the proliferative status of human hematopoietic progenitor cells.
Hum Gene Ther
1
1990
257

Author notes

Address reprint requests to Donald B. Kohn, MD, Mailstop #62, Childrens Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA 90027; e-mail:dkohn@chla.usc.edu.