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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2002-02-0599.
GENE THERAPY
From the Terry Fox Laboratory, British Columbia Cancer
Agency and University of British Columbia, Vancouver, BC, Canada;
Harvard-MIT, Division of Health Sciences and Technology,
Massachusetts Institute of Technology, Cambridge; Genetix
Pharmaceuticals, Cambridge, MA; and Albert Einstein College of
Medicine, Bronx, NY.
Transfer of therapeutic genes to human hematopoietic stem
cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein
(GFP) and a human Inherited disorders of More complex vectors have generally been less stable in viral producer
cells with the generation of subgenomic and rearranged forms and
typically low titers of useful virus. This has been particularly true
of Retroviral vector and packaging cell lines
Human cells
Transduction protocol and assessment of transduced cells
Aliquots of the recovered cells were then assessed by FACS directly
(fetal liver) or after being cultured for another 2 days (without
virus) in fresh serum-free medium containing the same growth factors as
were used in the prestimulation cultures to determine the proportion of
total and CD34+ GFP+ cells present. Other
aliquots were plated directly into colony-forming cell (CFC) and
long-term culture-initiating cell (LTC-IC) assays (see below) and the
proportion of GFP+ colonies obtained directly (or in the
6-week CFC assays of the LTCs) determined by direct visualization under
a fluorescent microscope. Additional aliquots were plated directly in
erythroid expansion cultures (see below) and the proportion of
propidium iodide (PI) Flow cytometry To determine the CD34+ cell content of starting cell populations and gene transfer efficiencies to the CD34+ cells present at the end of the infection protocols, cells were first incubated for 10 minutes at 4°C in cold Hanks balanced salt solution supplemented with 2% fetal calf serum (HF; StemCell Technologies) plus 5% pooled normal human serum (HF/5% HS), to block Fc receptors and minimize nonspecific binding of labeled antibodies. The cells were then labeled for 30 minutes at 4°C with antihuman CD34-fluorescein isothiocyanate (FITC)-8G1229 or CD34-Cy5 (for analysis of retrovirally transduced cells) and CD38-phycoerythrin (PE; Becton Dickinson, San Jose, CA) or antiglycophorin A-PE 10F7 antibody. Cell suspensions were then washed twice with HF, the last wash containing 1 µg/mL PI (Sigma Chemical) and analyzed on a FACSort (Becton Dickinson) using Cell Quest software (Becton Dickinson).Cells harvested from the marrow of immunodeficient mice that received
transplants of transduced cells (see below) were stained with antihuman
CD34-Cy5 plus CD19-PE and CD20-PE (Becton Dickinson) or antihuman
CD15-PE (Pharmingen, Mississauga, ON, Canada) or antihuman CD45-PE
(Becton Dickinson) and CD71-PE (OKT-9), or antihuman glycophorin A-PE
10F7. Cells harvested from the marrow of immunodeficient mice that
received transplants with manipulated but nontransduced human cells
were stained with antihuman CD34-FITC 8G12 plus CD19-PE and CD20-PE
(Becton Dickinson) or antihuman CD71-PE (OKT-9) and CD45-PE plus
CD15-FITC and CD66b-FITC (Pharmingen), or antihuman glycophorin-A-FITC
10F7 and Ter-119-PE (Pharmingen) to determine the proportion of mice
positive for these human populations. Positivity in all of these
analyses was defined as the presence in the PI In vitro progenitor assays The CFCs were assayed in methylcellulose medium (H4330; StemCell Technologies) supplemented with 50 ng/mL human Steel factor and 20 ng/mL IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF; Novartis), IL-6, G-CSF, and 3 U/mL human erythropoietin (Epo; StemCell Technologies) as described.30 Cells were assayed for LTC-ICs in 6-week cocultures containing mixed feeders of M2-10B4 and Sl/Sl murine fibroblasts genetically engineered to produce human IL-3 (10 ng/mL), G-CSF (130 ng/mL), and Steel factor (10 ng/mL).30 Fetal liver LTC-IC values were determined by dividing the total CFC output obtained from bulk culture assays by 72 based on previous experiments showing this to be the average CFC output of LTC-ICs from human fetal liver assayed in this way.24 Erythroid differentiation cultures were performed essentially as described31 by incubating the cells for the first 9 days in DMEM/plus 15% FCS plus 1 U/mL Epo, 100 ng/mL human SF, 40 ng/mL insulinlike growth factor-1 (IGF-1; R & D systems, Minneapolis,
MN), 10 6 M freshly dissolved hydrocortisone (Sigma),
106 M 17 -estradiol (Sigma), 1.28 µg/mL
iron-saturated transferrin (StemCell Technologies) and
104 M -mercaptoethanol, with half-media changes every
2 days. The cells were then harvested and cultured for another 11 days
in DMEM/plus 15% FCS, with 1 U/mL Epo, 1 µg/mL insulin
(Sigma), 1.28 µg/mL iron-saturated transferrin (StemCell
Technologies), and 104 M -mercaptoethanol, again with
half-media changes every 2 days.
Animals and in vivo studies NOD/LtSz-scid/scid (nonobese diabetic/severe combined immunodeficient [NOD/SCID]) mice32 and NOD/LtSz-scid/scid2-microglobulin null (NOD/SCID-2m/) mice33
were bred and maintained in microisolators in the animal facility of
the British Columbia Cancer Research Centre (Vancouver, BC). Six- to
8-week-old mice were sublethally irradiated with 350 cGy from a
137Cs source the day prior to being intravenously injected
with human cells. Mice were killed 6 weeks after transplantation and
cells flushed from the shafts of the 4 hind leg long bones using a
syringe and 21-gauge needle prefilled with HF/5% HS and a single-cell suspension obtained by gentle aspiration. Cells were stained and assessed for their content of human cells. The number of human cells of
a given phenotype per mouse was calculated by multiplying the percent
positive cells by 4 times the total number of cells harvested from 2 femurs and 2 tibias (based on the report that these 4 bones contain
approximately 25% of the total marrow mass of the adult
mouse34). The frequency of human fetal liver cells able to
repopulate sublethally irradiated NOD/SCID and
NOD/SCID-2m/ mice with both lymphoid and
myeloid cells was determined as previously described by limiting
dilution analysis of the proportions of mice injected with defined
numbers of cells that were not positive for both of these human
populations (ie, both CD34CD19/20+ cells and
CD45/71+CD 15/66b+ cells).35,36
Secondary transplantations were performed by harvesting cells from
primary NOD/SCID mice and transplanting 3.5 to 35 × 106
of their pooled bone marrow cells per secondary mouse (ie, 3.5%-22% of the total marrow of a primary mouse per secondary mouse based on the
assumption that 2 femurs and 2 tibias represent 25% of the total mouse
marrow).34 Secondary mice were 6- to 8-week-old sublethally irradiated NOD/SCID mice that were then killed for assessment of the presence of human cells in their marrow another 6 weeks later.
Reverse transcriptase-polymerase chain reaction analysis Total RNA was extracted using a commercial kit (Trizol, Gibco BRL) and reverse transcribed (RT) by random priming using 1 µg total RNA and superscript II reverse transcriptase (Gibco BRL) at 42°C for 30 minutes, followed by denaturation at 72°C for 10 minutes and snap cooling to 4°C for 5 minutes. A polymerase chain reaction (PCR) was then performed using primers specific for the retroviral-globin
gene (5'-GAG AAG TCC GCC GTT ACT GTT-3' and 3'-GAA GTT CTC AGG ATC CAC
GT-5') to amplify the expected 315-base pair (bp) fragment. After 40 cycles of denaturation (30 seconds at 94°C), annealing (30 seconds at
58°C), and extension (60 seconds 72°C), the PCR products were
separated on a 1.5% tris acetate EDTA (TAE) agarose gel and a
Southern blot performed using a -globin probe labeled with
32P by random priming.
Southern blot analysis Southern blot analysis was performed on the selected high producer clone of PG13 cells (clone H7 (PG13H7) using standard methods for DNA isolation and SStI or HindIII digestion. SstI cuts once in each LTR to release a 5.2-kb fragment encompassing the intact proviral sequence, and HindIII cuts once within the-globin gene insert to
detect unique fragments after incorporation of the vector into host
cell DNA (Figure 1A). The enhanced green fluorescent protein (EGFP)
gene was labeled with 32P by random priming and used as
a probe.
Efficiency of gene transfer of the MSCV-HS2- human fetal
liver cell preparations enriched for CD34+ or
CD34+CD38 cells were infected with the
MSCV-HS2--globin-GFP virus using the supernatant infection protocol
described in "Materials and methods." The frequency of gene
transfer to various types of progenitors detectable in vitro was then
assessed by measurements of GFP expression in their progeny. The
results are summarized in Table 1.
Although the proportions of all cells (and all CD34+ cells)
immediately after transduction that were GFP+ were 2-fold
higher in the cells that were initially selectively enriched for
CD34+CD38 cells (P < .05), both
target populations yielded similar frequencies (~55%) of
GFP+ colonies in the CFC assays of these cells. Similar
proportions (36%) of glycophorin A+ GFP+ cells
were also obtained in the 3-week erythroid differentiation cultures.
LTC-IC assays were performed only in the experiments using the fetal
liver cells that had been enriched for lin
CD34+CD38 cells. The transduction efficiency
measured on the colonies obtained from the 6-week colony assays of
these cells showed that a similar proportion (41%) of these primitive
progenitors had been transduced. Results of subsequent experiments
using lin cord blood cells (enriched for
CD34+ cells) were similar (Figure
2).
Efficiency of transfer of the MSCV-HS2- 2m/ mice and the presence of
GFP+ human cells assessed in the marrow of these mice 6 weeks later. The frequencies of different types of human cells detected
in mice injected with unselected fetal liver cells immediately after transduction are shown in Table 2.
Although absolute levels of engraftment were highly variable in both
types of immunodeficient hosts, significantly higher numbers of human
progeny were evident in the NOD/SCID-2m/
mice when the human cell outputs were normalized to the number of
CD34+ cells from which the transplant was derived (Table
2). On the other hand, the proportions of total human cells regenerated
in the 2 types of mice that were B lymphoid (CD19/20+)
versus granulopoietic (CD15+) or erythroid (glycophorin
A+) cells were not significantly different
(P > .15 and P > .35, respectively). In
mice repopulated with detectable numbers of GFP+ human
cells, these were consistently represented in all of these 3 lineages
of differentiating human cells. An example of the multilineage reconstitution observed in such mice is shown in Figure
3 (bottom panels). For comparison,
results obtained with mock-transduced cells are also shown (Figure 3,
top panels). As shown in Figure 4, when
the numbers of GFP+ human progeny in the B-lymphoid and
granulopoietic or erythroid compartments in individual recipients were
analyzed, these values could be seen to be significantly
correlated.
For these experiments each recipient mouse received transplants from
the transduced progeny of relatively large numbers of originally
lin
Because of the likelihood that most clinical gene therapy protocols for
treating human
Expression of the transduced human -globin gene expression directly
after harvest or after being placed in liquid erythroid expansion
cultures for 3 weeks as described in "Materials and methods." As
illustrated in Figure 6, about 95% of
all the cells obtained after 3 weeks under these conditions were
terminally differentiating human erythroid cells (glycophorin
A+ Ter 119) of which 35% were typically
still expressing GFP. Figure 7 shows a
representative Southern blot of the products of an RT-PCR reaction applied to RNA extracted from different sources of human cells generated from transduced fetal liver cells. Evidence of transcripts, albeit at low levels (estimated at < 1% of endogenous -globin messenger RNA) that could be amplified only from the transgene (Figure
1), was obtained both from cells harvested directly from mice
reconstituted with human cells (4 of 5 mice tested) and from cells
generated in 3-week erythroid expansion cultures (2 of 2 cases tested).
Interestingly, in one of the latter cases, the cells obtained from the
expansion culture were positive, whereas no transcripts were apparent
in the directly harvested cells.
This study represents part of a multistep effort to develop useful
vectors for the gene therapy of patients with hemoglobinopathies arising from mutations in the It is important to note, however, that the ease of obtaining benefit
from a gene therapy approach varies significantly from one disease
setting to another. In the case of SCID patients, even low levels of
transgene expression are sufficient to improve the disease and also to
confer a significant selective advantage on the growth of the lymphoid
progeny of the transduced HSCs. In contrast, for any We chose to use human fetal liver cells as the first source of primary
human target cells because this organ contains high numbers of
erythroid precursors as well as transplantable HSCs.42 Also we had recently found that human fetal liver HSCs generate much
higher numbers of erythroid progenitors in NOD/SCID mice than their
counterparts in cord blood or adult bone marrow,43 suggesting that their use might allow easier detection of transduced Nevertheless, we also found that the price of this efficient gene transfer protocol was a huge (90-fold) loss of the most primitive type of transplantable fetal liver HSCs currently detectable51 by the end of the 4- to 5-day in vitro transduction protocol. Some of this loss may be unavoidable resulting from repeated centrifugation of the cells or difficulties in harvesting all of the HSCs at the end of the infection procedure. On the other hand, it was reassuring to see that exposure to virus was not associated with any toxicity, because HSC losses were the same in mock-treated control samples. In fact, very few studies have quantitated the magnitude of the loss in HSCs that is widely known to occur during the prolonged retroviral infection protocols needed to attain the high levels of gene transfer to human HSCs reported by a number of groups.21,48,49,52 Moreover, these gene transfer efficiencies are always given for the HSCs present at the end of the transduction protocol and absolute yields of transduced HSCs are rarely reported. However, those that have indicate losses of cord blood HSCs in the order of 10- to 20-fold.53,54 The even greater losses measured here for transplantable fetal liver HSCs suggest that these HSCs are even more prone to death or differentiation in the presence of a growth factor combination that had been optimized for cord blood HSC transduction. It is interesting to note that, even in the mouse, defined culture conditions suitable for stimulating a net expansion of fetal liver HSCs in vitro have not yet been identified,55 in contrast to HSCs from adult marrow where a net expansion of these cells can be reproducibly achieved after 10 days in vitro.56 This suggests that during ontogeny, HSCs may undergo significant changes in the mechanism(s) they use to allow growth factor modulation of their continuing HSC status on being stimulated to divide. It was therefore important to establish that the same viral vector used
to transduce human fetal liver HSCs was also able to transduce human
cord blood progenitors that give rise to erythroid progeny in vivo. For
this we confined our studies to human myeloid-restricted short-term
repopulating cell targets that give a peak but transient output of
myeloid cells (including erythroid cells) 3 weeks after injection into
NOD/SCID- Taken together these experiments provide an important next step toward
the successful development gene therapy approaches to the treatment of
human
We thank the staff of the Stem Cell Assay Service for their
assistance in the initial preparation of primary human cell samples, Gayle Thornbury and Giovanna Cameron for operating the FACS, and Amy
Ahamed for secretarial assistance. We are also grateful to Amgen,
Cangene, Isolab, Novartis, Takara Biomedicals, and StemCell Technologies for valuable gifts of cells or reagents and Dr L. Shultz
for NOD/SCID
Submitted February 27, 2002; accepted March 29, 2002.
Prepublished online as Blood First Edition Paper, July 5, 2002; DOI: 10.1182/blood-2002-02-0599.
Supported by a grant from the National Institutes of Health (PO1 HL55435). I-H.O. held a Postdoctoral Fellowship from the National Cancer Institute of Canada (NCIC) and C.J.E. was a Terry Fox Cancer Research Scientist of the NCIC.
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.
Reprints: Connie J. Eaves, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, BC, V5Z 1L3, Canada; e-mail: ceaves{at}bccancer.bc.ca.
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Transduction of human CD34+CD38
© 2002 by The American Society of Hematology.
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  G. F. Atweh, J. DeSimone, Y. Saunthararajah, H. Fathallah, R. S. Weinberg, R. L. Nagel, M. E. Fabry, and R. J. Adams Hemoglobinopathies Hematology, January 1, 2003; 2003(1): 14 - 39. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-globin transgene in erythroid cells
derived from retrovirally transduced transplantable human fetal liver
and cord blood cells
Top
Abstract
Introduction
indicates the site
of the extended packaging signal. Panel B shows a Southern blot of
SstI-digested DNA (left) and HindIII-digested DNA
from the PG13 producer cell clone (H7) probed with a GFP probe. The
SstI blot shows a single band corresponding to the expected
full-length vector. The HindIII blot indicates that 5 copies
of the vector had been integrated into these producer cells.
) and 16 NOD/SCID-
). A similar plot
is shown on the right for GFP+ human CD19/20+
B-lymphoid cells numbers as a function of GFP+ human
glycophorin A+ erythroid cells in the same mice. Values
shown were calculated assuming that the content of 2 tibias and 2 femurs represents 25% of the total marrow mass of a
mouse.
