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GENE THERAPY
From the Department of Veterans Affairs Medical Center,
Reno, NV; Fleischmann Agriculture, University of Nevada, Reno, NV; and
Gene Therapy Laboratories, University of Southern California School of
Medicine, Norris Cancer Center, Los Angeles, CA.
The successful transduction of hematopoietic stem cells and
long-term (28 months) transgene expression within the hematopoietic system following the direct injection of high-titer retroviral vectors
into preimmune fetal sheep was previously demonstrated. The present
studies extended these analyses for 40 months postinjection and
evaluated whether the longevity of transgene expression in this model
system was the result of induction of prenatal tolerance to the
transgene product. The intraperitoneal injection of retroviral vectors
into preimmune sheep fetuses transduces thymic epithelial cells thought
to present antigen and thus define self during immune system
development. To directly demonstrate induction of tolerance, postnatal
sheep were boosted with purified To date, no postnatal hematopoietic stem cell
(HSC)-based gene therapy protocol has resulted in a cure or definitive
clinical improvement, with the exception of a recent case of severe
combined immunodeficiency disease1 in which the transduced
HSCs would be predicted to have a significant growth advantage over the
host's endogenous hematopoietic cells. While transduction efficiency of HSCs from humans and large animals in vitro has improved
dramatically with the development of newer generation vectors and a
better understanding of the in vitro growth requirements for HSCs, the level of gene-marked cells in vivo following transplantation of ex
vivo-transduced HSCs has remained well below the levels required for
clinical benefit.2-6 Interestingly, several studies have demonstrated that hematopoietic progenitors grown in vitro from the
patients are marked at a high enough frequency that some clinical improvement could be possible, yet the level of marked progeny in the
circulation has been so low as to be nearly undetectable except by
polymerase chain reaction (PCR).5,6 As yet, no explanation
has been offered for this discrepancy. While it is clear that
inefficient transduction of long-term repopulating HSCs and
transcriptional silencing of the vector promoters likely contribute to
the low levels of gene-marked cells within the circulation and the
transient nature of transgene expression, a large body of evidence is
emerging to suggest that host immune responses against both the vector
and the vector-encoded transgene products are a major limiting factor
to achieving successful genetic correction by gene
therapy.7-11 Studies have now shown that it is the host immune response and not transcriptional shutdown that precludes successful long-term transgene expression following adenoviral-mediated gene transfer.7-9 Other studies employing vectors based on
Moloney murine leukemia virus (MuLV), the backbone on which the vectors in most current clinical trials are based, have now shown that both the
vector itself and transgenes expressed from MuLV-based vectors are
capable of eliciting robust immune responses. McCormack et al showed
that intramuscular injection of MuLV-based retroviral vectors into both
adult mice and nonhuman primates elicited an anti-MuLV antibody
response that was able to neutralize vector-mediated transduction of
target cells in vitro.11 The authors then went on to
demonstrate that both immunoglobulin G and cytotoxic T-lymphocyte responses developed that were specific for the vector-encoded gene
products. In other studies employing an ex vivo transduction/stem cell
transplantation approach,12 Dube's group demonstrated
that the infusion of autologous cells transduced with retroviral
vectors encoding the normal canine Performing gene therapy in utero offers the possibility of correcting
genetic disorders prior to the onset of disease. In addition, there are
several features of the developing fetus that suggest that it may
represent a more amenable target for gene therapy than either the
neonate or the adult. One of these characteristics is the immunologic
naïveté and resultant permissive environment of the early
gestational fetus. These 2 characteristics should permit the acceptance
of cells and vector without the need for immunosuppression or
myeloablation. In early immunologic development, before thymic
processing of mature lymphocytes, the fetus appears to be receptive to
foreign antigens.15,16 Furthermore, exposure to foreign
antigens during this period often results in sustained tolerance, which
can become permanent if the presence of antigen is
maintained.17,18 We have previously reported that direct intraperitoneal injection of both low-titer and high-titer retroviral vectors into sheep fetuses during early gestation when the fetus is
still largely preimmune resulted in the successful transfer of the
transgene to primitive HSCs and the long-term persistence of
transgene-expressing hematopoietic cells in the circulation of the in
utero-transduced sheep.19,20 While in our previous studies with low-titer vectors we followed the animals for nearly 5 years after gene transfer, the follow-up studies with high-titer vector
only involved a 28-month analysis of the recipients. In the present
studies, we have extended the analyses of the animals receiving the
high-titer G1nBgSvNa8.1 vector (titer 1 × 107
colony-forming units [CFU]/mL) for 40 months postinjection and evaluated whether the longevity of transgene expression in this model
system was the result of induction of immune tolerance to the
vector-encoded transgene product following administration of the vector
during the period of immune-naïveté. We now demonstrate that transgene-expressing hematopoietic cells persist within the peripheral blood (PB) and bone marrow (BM) of these animals for at
least 40 months posttransduction. In addition, we show that the direct
injection of retroviral vectors into sheep fetuses during the period of
preimmunity results in the transduction of the epithelial cells that
are present in the fetal thymus and are responsible for presenting
antigen and defining self during immune system
development,21,22 showing that the groundwork for
induction of tolerance had been laid by this approach to gene transfer.
Direct evidence for induction of tolerance comes from experiments in
which control sheep and in utero-transduced sheep were boosted
postnatally with purified In utero gene transfer protocol
NeoR- and lacZ-specific
PCR/Southern blotting of PCR
products
Detection of transgene expression Expression of the NeoR and lacZ transgenes in PB and BM was evaluated using immunofluorescence microscopy and flow cytometric analysis and by performing hematopoietic progenitor assays in the presence and absence of a lethal concentration of G418, as we have previously described.19,20Isolation and culture of thymic epithelial cells Thymi were obtained from 1 normal control sheep and 3 in utero-transduced sheep at 76 days of gestation (21 days postinjection) and homogenized in Iscoves modified Dulbecco medium (Gibco Life Technologies, Rockville, MD) using a glass homogenizer to obtain a single-cell suspension. Cells from each thymus were then cultured until near confluence (about 2 weeks) in 2 gelatin-coated T75 flasks (Costar) in -minimal essential medium (Gibco) containing 10%
heat-inactivated fetal bovine serum (FBS), 0.5 µg/mL hydrocortisone, 20 ng/mL epidermal growth factor, 5 × 10 5 M
2-mercaptoethanol, and 5 × 109 M cholera toxin to
promote selective growth of epithelial cells.24 One
flask of cells from the control sheep was used as a control for the
specificity of x-gal staining. The other was used to assess the
susceptibility of fetal thymic epithelium to transduction with the
amphotropic MuLV vector administered in utero. To this end, one
subconfluent flask of control sheep thymic epithelium was transduced
for 72 hours with G1nBgSvNa8.1 supernatant, changing the supernatant
each 12 hours. Then, 48 hours after completion of the transduction,
G418 was added to the medium at a final concentration of 500 µg/mL
and the cells were grown to near confluence (about 1 week). One flask
of control thymic epithelium, in vitro-transduced thymic epithelium,
and thymic epithelium (grown in the absence of G418) from each of the
in utero-transduced sheep was stained with a pancytokeratin antibody
(Sigma Chemical, St Louis, MO) to confirm that the cultures were
composed purely of epithelial cells. When this had been confirmed,
another flask of thymic epithelium from each of these sources was then
evaluated for the expression of the lacZ transgene using a commercially
available x-gal staining system (Specialty Media, Lavallette, NJ).
Evaluation of immune tolerance Three experimental sheep and 3 control sheep were immunized subcutaneously at 6 separate sites with 50 µg of each of the following proteins: -gal (Boehringer Mannheim, Indianapolis, IN),
chicken egg albumin, and sheep albumin (both from Sigma). All primary immunizations were administered in complete Freund adjuvant, while reimmunizations (1 month after primary immunizations) employed incomplete Freund adjuvant (Sigma).
Assessment of humoral immune response to the transgene product Serum samples were collected at intervals for the next 2 months. Sera were tested for anti--gal antibody activities using a standard
enzyme-linked immunosorbent assay.25,26 Briefly, individual plates were coated (5 µg/well) with -gal, chicken egg
albumin, or sheep albumin, and nonspecific sites were blocked with
0.05% Tween 20, 2% bovine serum albumin in phosphate-buffered saline
(blocking buffer). Sera were added to the coated plates in serial
dilutions, incubated for 1 hour at 37°C, and rinsed 3 times with
blocking buffer following incubation. Donkey antisheep immunoglobulin G
antibody conjugated with peroxidase (Boehringer Mannheim) was added to
each well, and the plates were incubated for an additional hour at
37°C. Following incubation and washing, peroxidase substrate, ABTS
(2,2'-azino-di-[3-ethylbenzthiazoline sulfonate]) solution with
enhancer (Boehringer Mannheim), was added to each well and incubated at
room temperature until color (green) development was sufficient for
photometric detection (5-10 minutes). Samples were measured with the
Bio-Rad 3550-UV microplate reader (Bio-Rad, Hercules, CA) at 405 nm. A
reference reading was also taken at 490 nm and subtracted from the
readings obtained at 405 nm.
Assessment of cellular immune response to the vector/transgene products Cellular immune response was assessed as previously described12 with minor modifications. In short, at 45 months posttransduction, the 3 in utero-transduced sheep and 3 normal age-matched controls that had received subcutaneous immunizations with purified-gal were evaluated for the ability of their PB mononuclear
cells (PBMCs) to proliferate in response to either autologous BM
stromal cells or autologous BM stromal cells that were transduced in
vitro with the identical vector that had been administered in utero. BM
stromal cells were grown by plating out whole BM cells in standard
long-term marrow culture medium consisting of Iscoves modified Dulbecco medium (Gibco) with 12.5% FBS (Hyclone Laboratories, Logan UT), 12.5%
horse serum (Stem Cell Technologies), and 106 M
hydrocortisone (Sigma). At 48 hours after culture initiation, the
nonadherent fraction was removed and fresh media added. The stromal
cultures were maintained by twice-weekly half-volume media changes. At
confluence, stromal cells were trypsinized and replated at a 1:3
dilution. Twenty-four hours later, the media were replaced with
G1nBgSvNa8.1 vector supernatant supplemented with 4 µg/mL protamine
sulfate, and the stromal cells were transduced for 3 days, changing the
supernatant each 12 hours. Control untransduced stromal cells were
manipulated similarly to the transduced stromal cells with the
exception that fresh media were used in place of retroviral
supernatant. At 48 hours after the completion of the transduction
cycle, 500 µg/mL G418 was added to the stromal cell cultures that had
been transduced. Stromal cells were maintained by 1:3 dilutions when
confluent. At passage 4 to 5, the untransduced and transduced
G418-selected stromal cells were plated in 100 µL RPMI with 15% FBS
in 96-well plates and cultured to near confluence. Stromal cells were
then treated with mitomycin C (Sigma), washed 3 times, and the media
changed to RPMI with 5% autologous sheep serum. Autologous sheep PBMCs
were used as responder cells and resuspended in RPMI with 5%
autologous sheep serum. A total of 100 µL of responder cells was
added at a ratio of 1:5 stimulator-to-responder cells in triplicate to
each transduced and control stroma sample. Pokeweed mitogen (Gibco) was
added to 3 of the wells with cells from each sheep to serve as a
positive proliferation control, while media alone, PBMCs alone (from
each individual sheep), and stromal cells alone (from each individual
sheep) served as negative controls. Cells were incubated at 37°C for
72 hours, and bromodeoxyuridine (BrdU) labeling reagent was then added
and the cells were incubated for an additional 24 hours. BrdU
incorporation was then analyzed according to manufacturer's
instructions (Boehringer Mannheim) using the Bio-Rad microplate reader
at 450 nm with a reference wavelength of 690 nm.
Long-term presence and expression of exogenous genes in in utero-transduced animals As previously reported,20 16 preimmune sheep fetuses were injected intraperitoneally with 0.2 to 0.6 mL replication-defective MuLV-based retroviral supernatant. Fourteen of these recipients were available for analysis (2 were lost shortly after birth due to accidents at the farm). Proviral DNA and transgene expression were consistently detected in 12 of these animals throughout the 28-month course of our initial studies. At 40 months posttransduction, 9 of the 12 positive recipients from this study were still available for analysis (2 of the animals had been killed for tissue distribution studies, and 1 died as a result of tetanus). At this time, all 9 remaining in utero-transduced animals contained hematopoietic cells expressing the lacZ transgene product,-gal, in
PB (0.4%-5.1%) and BM (1.2%-5.3%) when evaluated by flow cytometry
using the fluorescent -gal substrate fluorescein
di--D-galactopyranoside (Table 1). In
addition, at 41 months posttransduction, all 8 animals that remained
(another animal was killed between 40 and 41 months for analysis of
vector tissue distribution) possessed neomycin phosphotransferase (NPT)
activity as demonstrated by the continued ability of hematopoietic
progenitors present in their BM to form significant numbers of
hematopoietic colonies in the presence of a lethal concentration (2 mg/L) of G418 in vitro (10.8%-37.0% G418 resistance).
NeoR-specific PCR analysis and subsequent Southern blotting
demonstrated that all G418-resistant colonies contained the bacterial
NeoR gene, confirming that vector-encoded
transgene expression was responsible for growth in a lethal
concentration of G418 (data not shown).
Expression of the transgene products was also demonstrated at 40 months
posttransduction by fluorescence-activated cell sorter (FACS) analysis
using antibodies specific for To confirm that proviral DNA had integrated into the host genome, lacZ- and NeoR-specific PCR analyses were performed on all in utero-transduced animals. Direct injection of retroviral vectors into the peritoneal cavity of fetal sheep resulted in the integration of proviral DNA (lacZ and NeoR genes) into PB and BM mononuclear cells of all in utero-transduced animals that persisted for at least 40 months posttransduction, suggesting that proviral DNA integration into primitive HSCs occurred following direct injection of retroviral vectors into preimmune fetal sheep (data not shown). Evaluation of immune tolerance to -gal. We next examined
whether our direct injection approach to in utero gene transfer
successfully transduced these cells in vivo within the recipient sheep.
To this end, we collected the thymi from 3 in utero-transduced sheep
at 21 days postinjection (76 days of gestation) and again grew pure
populations of epithelial cells from each thymus in the absence of G418
selection. The resultant monolayers were then subjected to x-gal
staining. As can be seen in Figure 1B, which is a representative flask
from one of the in utero-transduced sheep, numerous
-gal-expressing blue cells are present. No blue cells were seen in
thymic epithelial cultures grown from normal control fetal sheep (data
not shown). The epithelial origin of all thymic cell cultures was
confirmed by staining with a pancytokeratin antibody prior to analysis
for transgene expression (data not shown). These results demonstrate
the successful transduction of thymic epithelium in vivo within the
gene transfer recipients. In previous studies,19,20 we
have demonstrated the continued presence of proviral DNA within the
thymi of in utero-transduced sheep for at least 29 months
postinjection by PCR. In addition, throughout the entire 4-year course
of these studies we have consistently detected -gal+
cells when thymic sections from these sheep were analyzed by immunofluorescence with an anti--gal antibody (data not shown). These combined results led us to speculate that prolonged thymic expression of the transgene following our in utero approach to gene
therapy may allow for induction of immunologic tolerance.
To directly assess whether tolerance was induced in the in
utero-transduced animals, we examined the ability of 3 in
utero-transduced sheep (nos. 698, 703, and 704) and 3 normal
age-matched control sheep (nos. 7119, 7122, and 7124) to mount humoral
immune responses after postnatal immunization with purified
Potential cellular immune responses against the transgene
product/transduced cells in the in utero-transduced sheep were also evaluated by lymphocyte proliferation assays. Three experimental sheep
(nos. 698, 703, and 704) and 3 normal age-matched control sheep (nos.
7119, 7122, and 7124) were evaluated at 45 months posttransduction (3 months after the first
To confirm that the immunization with
We have previously reported the safe and efficient
transduction of primitive HSCs in sheep following direct injection of
retroviral vectors into the peritoneal cavity of preimmune
fetuses.19,20 In the present studies, we extended the time
course of our analysis and show that expression of the transgene has
persisted within the hematopoietic system of these in utero-transduced
animals for over 40 months posttransduction without eliciting an immune response to the vector-encoded genes. The long-term expression of the
immunogenic During early fetal development, there is a window of opportunity, prior to thymic processing of mature lymphocytes, during which the fetus is receptive to foreign antigens.15,16 Additionally, exposure of the fetus to foreign antigens during this window of opportunity results in sustained tolerance, which could be permanent if antigens are constantly maintained.17,18 Cellular tolerance appears to be dependent upon avidity thresholds and clonal deletion of reactive lymphocytes in the thymus, whereas the mechanism of B-lymphocyte tolerance (peripheral tolerance) appears to involve both clonal deletion and clonal suppression.21,22,41-45 The end result is an immune system that is tolerant to these specific foreign antigens. In the present studies, we administered the MuLV-based vector
supernatants into the peritoneal cavity of fetal sheep at days 55 to 60 of gestation. The corresponding period in humans is 13 to 14 weeks of
gestation, during which the human fetus is considered to be
immunologically naive. In sheep, the immune system reaches at least
partial maturity between days 67 and 77 of gestation as demonstrated by
the prolonged survival of allogeneic skin grafts placed before day 67 of gestation and vigorous rejection of grafts placed after day 77 of
gestation.46,47 We reasoned that by administering the
vector prior to immunocompetence, we could avoid the development of an
immune response to the vector/transgene products and thus achieve
long-term transgene expression. In addition, we felt that the delivery
of the vector at this early stage in development coupled with the
stable long-term expression of the transgenes afforded by the
MuLV-based vectors we employed might have induced tolerance to the
vector/transgene products. Our results demonstrate a complete absence
of an immune response to the vector-encoded Recent reports have demonstrated that tolerance to vectors and
transgene products could be induced if lymphocytes underwent thymic
selection in the presence of vector antigens48-52 and that this tolerance led to a prolongation of transgene expression. For
example, DeMatteo and colleagues48,49 showed that
intrathymic inoculation of neonatal mice with a recombinant adenoviral
vector encoding the lacZ gene during the period prior to T-cell
maturation resulted in the induction of host tolerance to the
adenoviral vector and the transgene product. Furthermore, when these
tolerized mice were injected intravenously as adults with the same
adenoviral vector, they exhibited an impaired adenovirus-specific
cytotoxic T-lymphocyte response, which allowed prolonged In conclusion, these studies demonstrate and confirm that the
direct injection of an engineered retroviral vector is a safe and
effective means of delivering exogenous genes to the primitive HSCs of
a developing fetus. We now also demonstrate that the long-term transgene expression obtained in this model is likely due to the induction of central tolerance to the transgene product,
Submitted November 9, 2000; accepted January 31, 2001.
Supported by grants HL40722, HL46566, and HL39875 from the National Institutes of Health and grant DK51427 from the Department of Veterans Affairs.
N.D.T. and C.D.P. contributed equally to this work.
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: Christopher D. Porada, V.A. Medical Center (151B), 1000 Locust St, Reno, NV 89502-2597; e-mail: porada{at}med.unr.edu.
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-galactosidase by in
utero gene transfer into preimmune sheep fetuses
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Abstract
Introduction
75°C with one Biomax intensifying screen.
) (nos. 7119, 7122, and 7124) and experimental (
) (nos. 698, 703, and 704) sheep.
Each value represents the mean ± SD. Each sample was assayed in
triplicate. Significant levels of anti-
