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Journal of Virology, February 2000, p. 1286-1295, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Marking and Gene Expression by a Lentivirus Vector
in Transplanted Human and Nonhuman Primate CD34+
Cells
Dong Sung
An,1
Robert P.
Wersto,2
Brian A.
Agricola,2
Mark E.
Metzger,2
Stephanie
Lu,1
Rafael G.
Amado,3
Irvin S. Y.
Chen,1,* and
Robert E.
Donahue2
Hematology Branch, National Heart, Lung, and
Blood Institute, Rockville, Maryland,2 and
UCLA AIDS Institute1 and
Department of Microbiology and Immunology and Molecular
Genetics and Department of Medicine,3 University
of California, Los Angeles, Los Angeles, California
Received 28 July 1999/Accepted 27 October 1999
 |
ABSTRACT |
Recently, gene delivery vectors based on human immunodeficiency
virus (HIV) have been developed as an alternative mode of gene
delivery. These vectors have a number of advantages, particularly in
regard to the ability to infect cells which are not actively dividing.
However, the use of vectors based on human immunodeficiency virus
raises a number of issues, not the least of which is safety; therefore,
further characterization of marking and gene expression in different
hematopoietic lineages in primate animal model systems is desirable. We
use two animal model systems for gene therapy to test the efficiency of
transduction and marking, as well as the safety of these vectors. The
first utilizes the rhesus animal model for cytokine-mobilized
autologous peripheral blood CD34+ cell transplantation. The
second uses the SCID-human (SCID-hu) thymus/liver chimeric graft animal
model useful specifically for human T-lymphoid progenitor cell
reconstitution. In the rhesus macaques, detectable levels of vector
were observed in granulocytes, lymphocytes, monocytes, and, in one
animal with the highest levels of marking, erythrocytes and platelets.
In transplanted SCID-hu mice, we directly compared marking and gene
expression of the lentivirus vector and a murine leukemia virus-derived
vector in thymocytes. Marking was observed at comparable levels, but
the lentivirus vector bearing an internal cytomegalovirus promoter expressed less efficiently than did the murine retroviral vector expressed from its own long terminal repeats. In assays for infectious HIV type 1 (HIV-1), no replication-competent HIV-1 was detected in
either animal model system. Thus, these results indicate that while
lentivirus vectors have no apparent deleterious effects and may have
advantages over murine retroviral vectors, further study of the
requirements for optimal use are warranted.
 |
INTRODUCTION |
A number of groups have recently
exploited the substantial available data regarding human
immunodeficiency virus type 1 (HIV-1) molecular biology and
pathogenesis to develop vehicles for gene delivery based on HIV-1 and
other lentiviruses (3, 9, 27, 30, 39, 40, 42, 43, 45, 47, 48, 51,
54). The simplest of these vectors consists of the minimal
cis-acting sequences required for HIV-1 replication. Other
HIV-1 genes are expressed in trans with a packaging plasmid.
The gene to be expressed, either a therapeutic gene or a reporter gene,
is expressed as part of an internal transcriptional unit inserted
between the long terminal repeats (LTRs) of the vector. Other
modifications of these vectors have resulted in the generation of
tat-dependent or inducible expression of the gene to be
expressed. A tat-dependent vector may be particularly
suitable for HIV-1 disease since it will be expressed only in the
presence of HIV-1 infection (5, 9). In some cases, HIV-1
vectors are themselves capable of inhibiting HIV-1 replication (5,
10, 15). Other therapeutic genes have been inserted into HIV-1
vectors, and it is likely that there will be increasing emphasis on the
potential use of these vectors for treatment of various human
conditions (22, 24, 29, 36, 52). The primary advantage of
lentivirus vectors is that by virtue of nuclear localization signals
present in HIV-1 proteins that are part of the preintegration complex
(11, 21, 26, 56), these vectors can efficiently infect some
nondividing cells (39, 41, 43, 48, 54), provided they reside
or progress through at least the G1b state of the cell
cycle (32). Other retroviral vectors based on murine
retroviruses require passage of the cell through mitosis in order to
integrate (34, 38, 49). Another advantage of lentivirus
vectors is that they have evolved to efficiently replicate in human
cells. However, the latter factor also underscores the need to
carefully assess the properties of lentivirus vectors, particularly
those derived from HIV-1, prior to use in humans.
Several animal model systems have been used to evaluate retroviral
vector delivery systems (2, 6, 8, 12, 13, 18, 41, 43). Of
particular relevance to lentivirus vector systems is the ability to
test transduction, reconstitution, gene expression, and marking and
ultimately therapeutic efficacy in model systems for human and/or
nonhuman primates. The rhesus macaque model system has been shown to be
amenable to transduction of CD34+ cells and transplantation
and is arguably the closest model system for human gene therapy
(7, 18, 20, 28, 53, 57). In addition, for some diseases such
as AIDS, the rhesus macaque will also allow for testing of therapeutic
efficacy against simian immunodeficiency virus (17, 23). Use
of this transplantation model, however, is expensive and does not lend
itself to the type of experimental manipulation required to test
multiple variables as do other small-animal model systems, such as the
SCID-NOD (50) and SCID-human (SCID-hu) chimeric mouse models
(37). In the SCID-NOD system, human CD34+ cells
are used to reconstitute an irradiated SCID mouse, resulting in the
production of human granulocytes, monocytes, and B cells (16,
25). In the SCID-hu system, human CD34+ cells can be
transplanted following irradiation of a chimeric human thymus/liver
(thy/liv) organ previously implanted into the mice to mimic human
thymopoiesis (2, 6, 8). Typically, the SCID-NOD mouse is
used to analyze non-T-lymphoid progenitor CD34+ cell
transplant, whereas the SCID-hu mouse is utilized to investigate T-lymphoid progenitor CD34+ cell transplant.
In this study, we used the rhesus macaque and SCID-hu system to
investigate the properties of transplanted CD34+ cells
transduced with a lentivirus vector bearing an internal cytomegalovirus
(CMV) promoter. We demonstrate that the lentivirus vector can result in
multilineage hematopoietic cell marking. However, comparative studies
using SCID-hu mice indicate that the levels of gene expression are
substantially lower than that of a murine retroviral vector using the
LTR as a promoter. We find no evidence for replication competent HIV-1
in either transplanted rhesus macaques or SCID-hu mice.
 |
MATERIALS AND METHODS |
HIV-1 vector construction and production.
An HIV-1-based
vector, pHR'-CMV-luciferase (43), was modified to contain
the enhanced green fluorescent protein (EGFP) cDNA with expression
driven by a CMV promoter (HR'CMVEGFP). A packaging plasmid for
the HIV-1-based vector, pCMVR8.2DVPR, was derived from pCMVR8.2
(43, 44) by deleting the vpr gene from nucleotide positions 5625 to 5731 by oligonucleotide-directed mutagenesis. Numbering of nucleotides starts at the 5' end of HIV-1 NL4-3 provirus (1). All vector stocks were generated by calcium
phosphate-mediated transfection of 293T cells (American Type Culture
Collection, Manassas, Va.). 293T cells were cultured in Dulbecco's
modified Eagle medium (DMEM) with 10% calf serum, 100 U of penicillin
per ml, and 100 µg of streptomycin per ml. 293T cells (2 × 107) were plated on 175-cm2 flasks in 25 ml of
the medium and transfected the following day with 5 µg of pHCMVG,
12.5 µg of pCMVR8.2DVPR, and 12.5 µg of HR'CMVEGFP for the
HIV-1-based vector. For the murine leukemia virus (MLV)-based vector
(SR
LEGFP with expression of EGFP from the 5' LTR), 5 µg of pHCMVG
(14), 12.5 µg of pSV
env
MLV
(33), and 12.5 µg of SR
LEGFP were used (5).
At 8 h posttransfection, the medium was replaced with 35 ml of
fresh medium. At 36 and 60 h posttransfection, the medium was
harvested, centrifuged at 1,500 rpm for 5 min (Sorvall RT 6000B; Ivan
Sorvall, Norwalk, Conn.), and filtered through a 0.45-µm-pore-size
filter. Further vector concentration was achieved by
ultracentrifugation at 50,000 × g for 90 min at 4°C.
The pellet was resuspended in Iscove's modified Dulbecco's medium
with 10% fetal calf serum (FCS), 100 U of penicillin per ml, and 100 µg of streptomycin per ml overnight at 4°C. The vectors were
concentrated 100-fold and kept in liquid nitrogen until use. Stocks of
vectors were titrated by infecting HeLa cells (105) with
various amounts of the virus and analyzing for EGFP expression by flow
cytometry on day 3 postinfection. The titers of vectors were
108 infectious units/ml for the HIV-1 vector and 2 × 107 infectious units/ml for the MLV-based vector.
Rhesus leukapheresis procedure.
Young adult rhesus macaques
(Macaca mulatta) that were serologically negative for simian
T-cell lymphotropic virus, simian immunodeficiency virus, simian
AIDS-related type D virus, and herpes B virus were used. Experimental
animals were quarantined and housed in accordance with federal
guidelines (44a) and the policies set by the Veterinary
Research Program of the National Institutes of Health. The protocols
were evaluated and approved by the Animal Care and Use Committee of the
National Heart, Lung, and Blood Institute.
Rhesus macaques received a combination of granulocyte
colony-stimulating factor (G-CSF; 10 µg/kg of body weight/day) and
stem cell factor (SCF; 200 µg/kg/day) (both kindly provided by Amgen, Inc., Thousand Oaks, Calif.) by subcutaneous injection for 4 days. Mobilized (i.e., cytokine-stimulated) leukapheresis cell product (mPB)
was collected from the peripheral blood by using a CS3000 Plus blood
cell separator (Baxter Healthcare, Fenwal Division, Deerfield, Ill.)
with a single, small-volume chamber and other modifications made to the
fluid path of the CS3000 Plus blood cell separator (19).
This allowed leukapheresis procedures to be performed on rhesus
macaques weighing <5 kg. Four rhesus macaques were transplanted. All
four animals received a total dose of 1,000 R of total-body
irradiation over 2 consecutive days prior to reinfusion of the
transduced cells. Peripheral blood was collected on the designated days
and evaluated by PCR analysis or flow cytometry for EGFP DNA or
expression, respectively.
Immunoselection of nonhuman primate CD34+ cells.
mPB CD34+ cells were isolated according to the
manufacturer's instructions by magnetic selection using a biotinylated
CD34+ antibody (clone 12.8; CellPro, Inc., Bothell, Wash.),
streptavidin MicroBeads, and MACS separation columns (Miltenyi Biotec,
Inc., Auburn, Calif.). Purity following the immunoselection procedure was routinely >95% as assessed by flow cytometry using an ELITE flow
cytometer (Beckman Coulter Corp., Miami, Fla.) after staining with an
allophycocyanin-conjugated anti-CD34 monoclonal antibody (MAb; clone
563; a kind gift from Gustav Gaudernack, Institution of Transplantation
Immunology, Rikshospitalet, The National Hospital, Oslo, Norway, with
allophycocyanin conjugation performed by Molecular Probes, Inc.,
Eugene, Oreg.).
Transduction of immunoselected rhesus macaque CD34+
cells.
Immunoselected CD34+ cells were transduced with
the vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1
vector (HR'CMVEGFP) either once or twice a day for 48 h for all
four animals on RetroNectin (BioWhittaker, Walkersville, Md.)-coated
non-tissue culture-treated six-well plates (Becton Dickinson Labware,
Franklin Lakes, N.J.) according to the manufacturer's instructions.
Transductions for two animals (95E008 and 95E009) were performed twice
a day in SCF alone (100 ng/ml) and 8 protamine sulfate (8 µg/ml); the
other two animals (RC504 and RC505) were transduced with the same HIV-1 vector only once a day at the same multiplicity of infection for the
48-h period in medium supplemented with SCF at 100 ng/ml, interleukin-6
(IL-6) at 50 ng/ml, and protamine sulfate at 8 µg/ml. The HIV-1
vector was used at a multiplicity of infection of approximately 5. EGFP
expression was measured by flow cytometry using a standard filter setup
for fluorescein (525-nm band-pass filter).
Quantitative PCR assay.
Each PCR amplification was performed
as described elsewhere (58). In brief, to detect HIV-1
vector sequences, one of the oligonucleotide primers for each pair used
was end labeled with 32P, and 25 ng was included in the
reaction (usually 5 × 106 to 1 × 107
cpm). The second oligonucleotide primer was not labeled, and 50 ng was
incorporated into each reaction. Each reaction mixture contained 0.25 mM each of the four deoxynucleoside triphosphates, 50 mM NaCl, 25 mM
Tris-HCl (pH 8.0), 5 mM MgCl2, 100 mg of bovine serum
albumin per ml, and 1.25 U of Taq DNA polymerase (Promega, Madison, Wis.). The reaction mixture was overlaid with 25 µl of mineral oil and then subjected to 25 cycles of denaturation for 1 min
at 94°C and polymerization for 2 min at 65°C. The reaction was
performed on a Perkin-Elmer thermocycler. Amplified products resulting
from the PCR were analyzed by electrophoresis on 6% nondenaturing
polyacrylamide gels and visualized by direct autoradiography of the
dried gels. Quantitative analysis of the amplified products was
performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.), and data were analyzed with the ImageQuaNT program (Molecular Dynamics). The nucleotide sequences of the oligonucleotide primers (M667 and AA55) used for HR'CMVEGFP DNA detection were derived from the nucleotide sequence of the HIV-1 LTR as previously described (58). A pair of oligonucleotide primers complementary to the first exon of the human
-globin gene (58) was used in
each reaction mixture in PCR analyses to normalize the total amount of
rhesus macaque cellular DNA present. During PCR amplification, labeled
-globin-specific oligonucleotides were incorporated into the
reaction at 5 × 106 to 1 × 107 cpm.
Quantitation of HIV-1 vector DNA during PCR amplifications was
performed by analyzing a standard curve of dilution of HR'CMVEGFP
plasmid DNA digested with
HindIII, which does not cleave
vector
sequences. This DNA was diluted in 0.01 µg of rhesus macaque
peripheral
blood mononuclear cell (PBMN) DNA per ml. The copy number of
HIV-1
vector included in the standard curve ranged from 3 to 1,000.
Standard curves for rhesus macaque

-globin DNA were obtained
by
amplification of 0.001 to 0.3 µg of rhesus macaque cellular
DNA (100 to 30,000 cell equivalents) from rhesus macaque
PBMN.
Transduction and immunoselection of gene-transduced human fetal
liver-derived CD34+ cells by flow cytometry.
Human
fetal liver-derived CD34+ cells were purified from a fetal
liver as previously described (6). Cells were cultured in
Iscove's modified Dulbecco's medium with 100 ng each of IL-3, IL-6,
and SCF (kindly supplied by Amgen) per ml, 20% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Fetal liver-derived CD34+ cells (2 × 106) were transduced
with VSV-G-pseudotyped HR'CMVEGFP or SR
LEGFP vector by incubating 2 ml of a 40-fold dilution of the HR'CMVEGFP vector stock or 10-fold
dilution of the SR
LEGFP vector stock in the presence of Polybrene (8 µg/ml) at 37°C for 2 h on day 1 or 2 after CD34+
cell purification from fetal liver, respectively. On day 3 after CD34+ cell purification from fetal liver, vector-transduced
CD34+ cells were stained with a MAb to human CD34 (Becton
Dickinson, Mountain View, Calif.) conjugated with phycoerythrin. EGFP
and CD34+ cells were sorted on a FACStarplus
(Becton Dickinson). Thy/liv implants of irradiated (300 rads) animals
were directly injected with 105 EGFP and CD34+
cells on day 4 after CD34+ cell purification from fetal liver.
Flow cytometric analysis for human thymocytes.
Thymocytes
were obtained by biopsy from reconstituted thy/liv implants of SCID-hu
mice 4 weeks postreconstitution. Thymocytes were stained with a MAb to
human CD1, CD3, CD4, CD5 CD8, or CD45 directly conjugated with
phycoerythrin or periodinin chlorophyll protein (Becton Dickinson).
Samples were run on a FACScan flow cytometer, and data analyzed with
the CellQuest program (Becton Dickinson). Ten thousand events were
acquired for analysis.
Cell culture for in vitro activation studies.
Human
thymocytes and rhesus macaque PBMN were cultured at a concentration of
106/ml in flat-bottom culture plates. Culture plates were
coated with goat anti-mouse immunoglobulin G (GAM; Tago, Burlingame, Calif.). GAM (10 g/ml) in phosphate-buffered saline (PBS; pH 7.4) was
added to wells and incubated for 2 h at 37°C. Plates were then
washed three times with PBS. Anti-human CD3 MAb (T3; 4 mg/ml in PBS;
Coulter, Hialeah, Fla.) or anti-monkey CD3 MAb (1 mg/ml; BioSource
International, Camarillo, Calif.) was added on to the GAM-coated
culture plates and incubated at 37°C for 1 h. The immobilized GAM provides a solid phase for binding of anti-human or monkey CD3 MAb
as previously described (35, 55). After washing, thymocytes obtained from thy/liv implants of SCID-hu mice or rhesus macaque PBMN
were cultured in the presence of IL-2 (10 U/ml; Amgen), anti-human CD28
MAb, and immobilized anti-human CD3 MAb or anti-monkey CD3 MAb in
Iscove modified Dulbecco's medium-20% FCS supplemented with
penicillin (100 U/ml) and streptomycin (100 mg/ml), respectively. For
control, cells were cultured in parallel in the absence of IL-2,
anti-CD28 MAb, and immobilized anti-CD3 MAb. After 3 days of
stimulation in vitro, thymocytes were analyzed by flow cytometry for
EGFP expression. [3H]thymidine incorporation was measured
by pulse-labeling cells for 6 h on day 2 as previously described
(58).
Coculture of rhesus macaque PBMN and activated human PBL.
PBMN (107) from lentivirus vector-transduced or
mock-transduced rhesus macaques were cocultured with 107
phytohemagglutinin (PHA)-activated human peripheral blood lymphocytes (PBL) in RPMI with 10% FCS and human IL-2 (10 U/ml). As a control, 107 PHA-activated human PBL infected with 1 ng of HIV-1
NL4-3 were cocultured with 107 PHA-activated human PBL.
Every 7 days, 107 activated human PBL were added to the
culture. At 4 weeks postcocultivation, culture supernatants were
harvested, measured for p24 by enzyme-linked immunosorbent assay
(ELISA), and subjected to MAGI cell assay.
MAGI assay.
The CD4-positive LTR-galactosidase-expressing
HeLa (MAGI) cell indicator line (31) was obtained from the
AIDS Research and Reference Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases, and maintained in DMEM
supplemented with 10% calf serum, 100 U of penicillin G sodium per ml,
0.1 mg of streptomycin sulfate per ml, 0.15 mg of G418 sulfate per ml,
and 0.1 mg of hygromycin B per ml. Cells were seeded at a density of
8 × 104 per well in a 12-well plate 24 h prior
to infection. Cell coculture supernatants of rhesus macaque PBMN and
activated human PBL (300 µl) were adsorbed to cells in the presence
of Polybrene (10 µg/ml) for 2 h at 37°C prior to the addition
of 1 ml of medium. As controls, culture supernatant (300 µl of p24
[1,303 ng/ml] of HIV-1 NL4-3-infected human PBL and uninfected
culture supernatant of MAGI cells (300 µl) were used. Following
incubation for 2 days at 37°C, the cells were fixed and stained with
5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) as
previously described (31).
Direct injection of HIV-1 vector into thy/liv implant of SCID-hu
mouse.
Prior to vector injection, SCID-hu thy/liv mice were
irradiated (200 rads). At 24 h (implants a and b) or 4 days
(implants c and d) postirradiation, 100-fold-concentrated HIV-1 vector
was directly injected into irradiated thy/liv implants of SCID-hu mice.
Approximately 50 µl of the vector stock was injected in each
irradiated implant. The titer of the HIV-1 vector was 108
infectious units/ml.
 |
RESULTS |
Transduction and transplantation of immunoselected mPB
CD34+ cells in rhesus macaques.
We used an HIV-1
vector bearing an internal CMV immediate-early promoter for expression
of the gene encoding EGFP. We evaluated this HIV-1 vector in the rhesus
macaque transplantation model. Nonhuman primate immunoselected
hematopoietic growth factor mPB CD34+ cells were transduced
on non-tissue-coated six-well plates treated with the recombinant
fibronectin fragment CH-296 (RetroNectin) either once or twice a day
for 2 consecutive days following collection. Because lentivirus vectors
can transduce nonmitotic cells, the CD34+ cells were
stimulated with combinations of cytokines (SCF alone or SCF plus IL-6)
that induced less cell division than combinations typically used and
may therefore induce less lineage-specific differentiation. On average,
7.6% (range, 3.2 to 13.2%) of the CD34+ cells expressed
EGFP following transduction with the lentivirus vector (Table
1). This transduction efficiency is
superior to that observed for murine retroviral vectors, as most cells
cultured under these conditions at various time points are not yet
susceptible to murine retroviral infection (data not shown).
Presence and expression of vector in rhesus PBMN
subpopulations.
Four animals were transplanted with autologous
CD34+ cells transduced with the HIV-1 vector. All animals
had uneventful hematopoietic reconstitution following total-body
irradiation and autologous transplant, with leukocyte counts returning
to 1,000 cells/µl within 15 days of transplant and platelet counts
returning to greater than 50,000/µl within 11 days of transplant
(Table 1). We monitored the presence of vector in different
hematopoietic lineages by fluorescence-activated cell sorting followed
by PCR analysis as well as by direct flow cytometric analysis for EGFP in gated cell populations. As determined by PCR analysis, approximately 0.1 to 1% of circulating leukocytes contained vector DNA in sorted lymphocytic and granulocytic cell fractions 10 weeks (animal 95E008) or
16 weeks (animal RC505) following transplant (Fig.
1). In one animal, RC505, we observed a
higher level of vector DNA in the granulocyte subpopulation.

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FIG. 1.
PCR analysis of rhesus macaque cell fractions following
transplant. DNA from rhesus macaque granulocytes, lymphocytes, and
whole PBMN were analyzed for HIV-1 vector transduction by PCR 10 weeks
after reconstitution of rhesus macaque 95E008 and 16 weeks after
reconstitution of rhesus macaque RC505. Granulocyte and lymphocyte
populations were sorted from PBMN to, respectively, 98 and 99% purity,
based on forward and side scatter. HIV-1 vector DNA-specific signal was
compared with that of amplified -globin DNA sequences to determine
the number of vector copies per cell (%HIV-1 vector DNA copies/cell,
calculated as number of HIV-1 vector DNA copies/number of cell
equivalents × 1/10 × 100). For PCR amplification, 10-fold
less DNA was used for -globin DNA PCR in order to obtain
quantitative -globin DNA signals. Quantitative HIV-1 vector DNA and
-globin DNA standards (std) were assayed along with DNA from a
nontransduced rhesus (negative control) in parallel. No signals were
detected for the negative control (data not shown). Percent EGFP
expression was determined by flow cytometric analysis at the same time
point as PCR. tRNA served as the negative control for the PCR assay.
|
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EGFP expression was detected in PBMN beginning at 2 weeks after
transplant. EGFP expression at 30 weeks posttransplant in
various PBMN
subpopulations is shown in Fig.
2. EGFP
is readily
detected in granulocyte, monocyte, and lymphocyte
subpopulations.
As determined by the mean fluorescence intensity, the
expression
in lymphocytes was the least efficient. Further analysis
indicated
that both B and T lymphocytes (CD20 and CD2 positive,
respectively)
as well as NK cells (CD16 CD56 double positive) expressed
the
EGFP gene (data not shown). The percent of cells expressing EGFP
was monitored over a period of 34 to 39 weeks (Fig.
3) in each
of four transplanted rhesus
macaques. EGFP expression was observed
in granulocyte, lymphocyte, and
monocyte subsets. Although there
was variation in the level of marking
over time, we observed a
general increase in marking between 5 to 10 weeks following transplant.
In the animal (RC505) with the highest
levels of marking, the
proportions increased over time to between 1 to
2% of PBMN. To
date, 14 months following transplant, the proportion of
PBMN expressing
EGFP has remained relatively stable, with the highest
levels ranging
from 1 to 3%.

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FIG. 2.
Flow cytometric analysis of rhesus macaque PBMC at 30 weeks. Circulating leukocytes at 30 weeks were evaluated by flow
cytometry in animals transduced with the HIV-1 vector (HR'CMVEGFP).
Granulocytes (GRAN), monocytes (MONO), lymphocytes (LYM), erythrocytes
(RBC), and platelets (PLAT) were gated according to size (forward
scatter) and granularity (log 90° scatter) and analyzed for EGFP
expression. Results for HIV-1 vector-transduced rhesus macaques RC505
and 95E008 are shown. EGFP expression was detected by flow cytometry in
all the subpopulations in RC505. The other three transplanted animals
had EGFP expression in granulocytes, monocytes, and lymphocyte
populations but did not demonstrate any detectable fluorescence in
erythrocytes and platelets. Data for rhesus macaque 95E008 are shown.
The x axis is log fluorescent intensity of EGFP; the
y axis represents the gated population based on forward and
side scatter (logarithmic fluorescence intensity).
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FIG. 3.
Kinetics of EGFP expression following transplant. The
percentage of EGFP-expressing granulocytes ( ), lymphocytes ( ),
and monocytes ( ) was determined at various time points as described
in the legend to Fig. 2 for all four animals receiving the
lentivirus-transduced immunoselected CD34+ cells. The
percentage of EGFP-expressing cells is shown over a 30- to 40-week
evaluation period.
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EGFP expression in erythrocytes and platelets.
Of particular
interest is the observation that low but significant levels of EGFP
could be detected 30 weeks posttransplant in the erythrocytes and
platelets of one animal, RC505, with the highest overall proportion of
marked PBMN (Fig. 2). Marking of these cell types has not previously
been observed in rhesus macaques transplanted with CD34+
cells transduced with a murine retrovirus (murine stem cell virus [MSCV]-based) vector (46). Since the majority of these
cells would not be expected to harbor vector DNA, we presume that we were detecting persisting EGFP protein. We cannot formally exclude the
possibility that we were detecting EGFP only in nucleated precursor
cells. However, this appears unlikely since if it were the case, the
majority of circulating erythroid precursor cells would have to express
vector, inconsistent with the frequency of marked cells in the other
hematopoietic lineages.
Activation of rhesus PBMN does not enhance the levels of vector
expression.
The mean fluorescence intensity of EGFP in lymphocytes
was about fivefold weaker than that in granulocytes (Fig. 2). We tested whether activation of PBMN cells with IL-2 and anti-CD3 MAb could enhance expression. Treatment of rhesus PBMN with IL-2 and anti-CD3 resulted in an approximately 100-fold increase in
[3H]thymidine incorporation. Activation of PBMN did not
appear to significantly increase the proportion of cells expressing
EGFP (Table 2) or EGFP fluorescence
intensity (data not shown).
Replication-competent HIV-1 is not evident.
To date, more than
50 weeks posttransplant, all four animals remain healthy and have not
demonstrated circulating antibody to HIV p24 by Western blotting,
circulating p24 antigen by ELISA, or evidence of circulating HIV-1
virus by quantitative HIV-1 ultrasensitive reverse transcription-PCR
(data not shown). We further confirmed that there was no evidence
of replication-competent or latent HIV-1 in the cells by coculture of
107 rhesus macaque PBMN with activated human PBL followed
by passage and repeated coculture for 4 weeks. The supernatant was then
assayed for HIV-1 p24 by ELISA and for replication-competent HIV-1 by MAGI assay. No evidence for replication-competent HIV-1 was found (Table 3).
HIV-1 vector-transduced CD34+ cells can reconstitute
irradiated SCID-hu thy/liv grafts.
We previously used human
thy/liv chimeric grafts transplanted into SCID-hu mice as a model for
progenitor cell gene therapy using murine retroviral vectors (2,
6). This small-animal model system allows the investigation of
marking of T-cell lineages in the human thymus throughout thymopoiesis,
not easily addressed experimentally in the rhesus macaque model.
SCID-hu thy/liv chimeric grafts transplanted 3 to 4 months previously
were irradiated and injected with 5 × 105 human fetal
liver CD34+ cells transduced with an HIV-1 or MLV-based
vector (SR
LEGFP). Both the HIV-1 and MLV vector-transduced
CD34+ cells can reconstitute SCID-hu thy/liv mice (Fig.
4). At 4 weeks after
introduction of CD34+ cells into thy/liv implants, the
percentage of EGFP-expressing cells ranged from 0.9 to 28% for HIV-1
vector-transduced implants and from 1.2 to 39% for MLV
vector-transduced implants (Fig. 4A). We confirmed that the
EGFP-expressing cells were human cells by using anti-CD45, a marker for
human lymphocytes. Three-color flow cytometric analysis determined that
when thymocytes were first gated on EGFP-positive cells and
subsequently analyzed for expression of CD4 and CD8, the distribution
of CD4+ and CD8+ cells for HIV-1 or MLV
vector-transduced cells was similar to that for mock-infected cells,
indicating that progenitor cells common to CD4+ and
CD8+ cell lineages were likely to have been transduced with
both vectors (Fig. 4B, compare d and e with c).

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FIG. 4.
(A) EGFP expression 4 weeks after reconstitution in
human CD45+ thymocytes of reconstituted thy/liv implants in
SCID-hu mice. EGFP expression at 28, 14, and 0.9% was detected in
human CD45+ thymocytes in three SCID-hu mice receiving a
thy/liv transplant transduced with the HIV-based vector and at 39, 11, and 1.2% in three SCID-hu mice receiving a thy/liv transplant
transduced with an MLV-based vector 4 weeks postimplant. Less than
0.2% of EGFP expression was detected in thymocytes from a SCID-hu
mouse receiving nontransduced cells as a thy/liv implant (data not
shown). Ten thousand events were analyzed. (B) Distribution of EGFP-expressing thymocytes 4 weeks after reconstitution of
thy/liv implants in SCID-hu mice. At 4 weeks after injection of human
EGFP+ CD34+ cells, thymocytes from
gene-transduced and reconstituted thy/liv implants were obtained by
biopsy of SCID-hu mice. Obtained thymocytes were stained for human CD4
and CD8 markers and evaluated for EGFP expression by flow cytometric
analysis. As shown in the lower panels, thymocytes found to express
EGFP were gated and analyzed for CD4 and CD8 distribution. EGFP
expression was detected in both CD4 and CD8 single-positive cells as
well as CD4 CD8 double-positive and CD4 CD8 double-negative thymocytes.
EGFP-expressing thymocytes showed a CD4 and CD8 profile similar to that
of non-gene-transduced reconstituted implant controls (mock). Ten
thousand events were analyzed.
|
|
The MLV vector resulted in gene expression higher than that of the
HIV-1 based vector.
In comparing the levels of EGFP expression for
the MLV and HIV-1 vectors in transduced thymocytes, we noted that the
HIV-1 vector expressed at levels of fluorescence intensity
approximately 10-fold-lower than that of the MLV vector. This
difference was consistent among reconstituted implants (Fig. 4).
Although we did not directly compare MLV and HIV-1 vector expression in
rhesus macaques, the fluorescence intensity of the HIV-1 vector in
SCID-hu thymocytes is similar to the fluorescence intensity in PBL in the rhesus macaques when analyzed with similar flow cytometric settings
(data not shown).
Activation of thymocytes does not induce increased level of EGFP
expression.
In transplanted rhesus macaques, we found that EGFP
expression could not be further induced following activation of PBMN
(see above). We similarly tested whether we could induce EGFP
expression following activation of the thymocytes. Thymocytes from
transplanted SCID-hu mice were stimulated in vitro with IL-2 and with
anti-CD3 and anti-CD28 MAbs for 3 days. The rate of thymidine
incorporation of the stimulated cells increased approximately 100-fold
over that of nonstimulated cells. The level of EGFP expression,
however, was only slightly induced following activation with anti-CD3
and IL-2 (Table 4). These results are
consistent with our previously published studies with an MLV vector
(6).
The HIV-1 vector is expressed throughout thymopoiesis.
We
previously demonstrated that a murine retroviral vector used to
transduce human CD34+ cells and transplanted to SCID-hu
mice was expressed in various thymocyte subpopulations
throughout thymopoiesis (6). We assessed whether gene
expression directed by the HIV-1 vector was similarly expressed. For
these studies, we modified the transduction and transplant protocol by
using a new and more rapid approach to assess the HIV-1 vector. The
vector was directly injected into irradiated thy/liv implants of
SCID-hu mice that had been irradiated to kill resident thymocytes and
induce progenitor cell function. At 4 weeks after transplant of the
transduced CD34+ cells into thy/liv implants, EGFP
expression was detected in CD45+ thymocyte populations
(Fig. 5A). Similar percentages of
EGFP-expressing cells were detected in all thymocyte subpopulations
tested (CD1, CD3, CD4, CD5, and CD8), indicating vector expression
throughout thymopoiesis (Fig. 5B). These results are consistent with
that previously observed for the MLV vector (6).

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|
FIG. 5.
EGFP expression in human thymocytes 4 weeks after direct
injection of HIV-1 vector in thy/liv implants of SCID-hu mice. Prior to
vector injection, SCID-hu thy/liv mice were irradiated (200 rads) to
induce progenitor cell proliferation and to kill resident thymocytes.
At 24 h (implants a and b) or 4 days (implants c and d)
postirradiation, 100-fold-concentrated HIV-1 vector was directly
injected into irradiated thy/liv implants of SCID-hu mouse. Four
irradiated implants (a through d) were each injected with approximately
50 µl of the vector stock. The titer of HIV-1 vector was
108 infectious units/ml. (A) At 4 weeks after direct
injection of HIV-1 vector into thy/liv implants, EGFP expression was
analyzed in human CD45+ thymocyte populations by flow
cytometry. (B) EGFP expression was analyzed in human CD1, CD3, CD4,
CD5, and CD8 thymocyte subpopulations. Representative data from implant
a are shown.
|
|
Reconstituted SCID-hu implants did not show any deleterious
effects.
Human thymocytes of the SCID-hu thy/liv mouse are highly
susceptible to death when challenged with infectious
replication-competent HIV-1. No toxic effects were observed in those
implants receiving the HIV-1 or MLV vector-transduced CD34+
cells. The percentages of CD4+, CD8+, and
CD4+ CD8+ cells expressing EGFP remained
similar to that of both mock-transduced and MLV vector-transduced
animals (Fig. 4B). Thus, no HIV-1-associated pathogenicity was observed
in the transduced animals, indicating that no replication-competent
HIV-1 was present in the viral stocks or generated after transduction,
consistent with the results observed in transplanted rhesus macaques.
 |
DISCUSSION |
Our results demonstrate that successful transplantation and
marking are obtained in rhesus macaque and SCID-hu mice that have been transplanted with nonhuman and human CD34+
cells, respectively, transduced with an HIV-1-based lentivirus vector.
Infusion of myeloablated rhesus macaques resulted in reconstitution and
marking of lymphoid, myeloid, and granulocyte lineages in all animals.
In the case of one animal with the greatest overall levels of marking,
EGFP expression was observed in erythrocytes and platelets, not
previously observed with MLV vectors (46). Parallel
experiments conducted with human CD34+ cells in SCID-hu
mice demonstrated reconstitution of thymopoiesis and evidence of vector
throughout various stages of T-cell differentiation. Other
investigators have demonstrated multilineage marking in a SCID-NOD
model system (41); however, that model system did not
examine T-lineage gene transfer and marking, critical for evaluation of
efficacy and potential deleterious effects of a vector derived from
HIV-1.
Gene expression in different cell types is dependent on the relative
strength of promoters used. The HIV-1 vector uses an internal CMV
promoter, whereas the murine retroviral vector uses the vector LTR.
HIV-1 vector expression of EGFP as monitored by the mean fluorescence
intensity in lymphocyte subpopulations was relatively weak. This level
of expression was at least 10-fold lower than that for the MLV-based
vector in SCID-hu thymocytes. Although rhesus macaque lymphocytes were
marked, only a low fluorescence intensity of expression from the HIV-1
vector was detected following transplant, about fivefold lower than
expression in rhesus macaque granulocytes, consistent with the lower
level of expression observed in the SCID-hu thy/liv thymocytes. For
both human and rhesus macaque lymphoid populations, no increase in
expression was observed following ex vivo T-cell activation.
We cannot say with certainty whether we have successfully transduced in
rhesus macaques a pluripotent hematopoietic stem cell, but several
lines of evidence suggest that the HIV-1 vector may have transduced an
early progenitor cell prior to commitment to the myeloid or lymphoid
pathway. First, the relative degrees of marking for both lymphoid and
granulocyte compartments are similar for each rhesus macaque as
determined by PCR. Second, these relative proportions have either
remained stable or even increased in certain instances 30 weeks
following reconstitution (data not shown). Third, in the animal with
highest overall levels of marking, EGFP expression could be observed in
multiple hematopoietic lineages, including erythrocytes and platelets.
Additional studies, however, will be required to improve transduction
efficiency and the level of EGFP expression in desired subpopulations
of hematopoietic cells.
Concerns have been raised regarding the use of vectors derived from
HIV-1 in humans (4). Since these vectors are defective for
HIV-1 envelope and generated by cotransfection with a VSV-G-expressing envelope vector, it is highly unlikely that a replication-competent virus could be formed from recombination between members of two distinct families of viruses. Nevertheless, we formally showed in both
transplanted rhesus macaques and SCID-hu mice that
replication-competent HIV-1 did not result. Therefore, we believe that
lentivirus vectors in principle are suitable for use in humans;
however, further refinements in vector design including enhanced
expression will be necessary before lentivirus vectors can be used
effectively for gene delivery.
 |
ACKNOWLEDGMENTS |
We thank Barrington Thompson, Earl West, the staff of Bionetics,
Inc., the Laboratory of Small Animal Surgery and Medicine, and the
staff of Jerry Zack's laboratory for assistance in caring for the
animals, and we thank Frances Ngok for technical assistance. We also
thank Liz Duarte for assistance in preparing the manuscript.
This work was supported in part by the UCLA CFAR and NIH grants AI39975
and AI36555.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: 10833 LeConte
Ave., 11-934 Factor Building, Los Angeles, CA 90095-1678. Phone: (310) 825-4793. Fax: (310) 794-7682. E-mail: rtaweesu{at}ucla.edu.
 |
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Journal of Virology, February 2000, p. 1286-1295, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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