|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1895-1905
Genetically Corrected Autologous Stem Cells Engraft, But Host Immune
Responses Limit Their Utility in Canine  -L-iduronidase Deficiency
By
Carolyn Lutzko,
Stephen Kruth,
Anthony C.G. Abrams-Ogg,
Kathy Lau,
Liheng Li,
Brian R. Clark,
Christine Ruedy,
Shaherose Nanji,
Robert Foster,
Donald Kohn,
Robert Shull, and
Ian D. Dubé
From the Department of Laboratory Medicine, Sunnybrook Health Science
Centre, and the Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario, Canada; the Department of
Clinical Studies, Ontario Veterinary College, University of Guelph,
Guelph, Ontario, Canada; the Oncology Research Program, Toronto
Hospital, Toronto, Ontario, Canada; Research Immunology and Bone Marrow
Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA; and
the Department of Pathology, College of Veterinary Medicine, University
of Tennessee, Knoxville, TN.
 |
ABSTRACT |
Canine  -L-iduronidase (-ID) deficiency, a model of the human
storage disorder mucopolysaccharidosis type I (MPS I), is an ideal
system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells. We performed adoptive transfer of
genetically corrected autologous hematopoietic cells in dogs with
-ID deficiency. Large volume marrow collections were performed on
five -ID-deficient dogs. Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks
of culture to retroviral vectors bearing the normal canine -ID cDNA.
Transduced LTMC cells from deficient dogs expressed enzymatically
active -ID at 10 to 200 times the levels seen in normal dogs. An
average of 32% of LTMC-derived clonogenic hematopoietic cells were
provirus positive by polymerase chain reaction and about
half of these expressed -ID. Approximately 107
autologous gene-modified LTMC cells/kg were infused into
nonmyeloablated recipients. Proviral DNA was detected in up to 10% of
individual marrow-derived hematopoietic colonies and in 0.01% to 1%
of blood and marrow leukocytes at up to 2 to 3 years postinfusion.
Despite good evidence for engraftment of provirally marked cells,
neither -ID enzyme nor -ID transcripts were detected in any dog.
We evaluated immune responses against -ID and transduced cells. Humoral responses to -ID and serum components of the culture media
(fetal bovine and horse sera and bovine serum albumin) were identified
by enzyme-linked immunosorbent assay. Cellular immune responses to autologous -ID but not neor transduced
cells were demonstrated by lymphocyte proliferation assays. To abrogate
potential immune phenomena, four affected dogs received posttransplant
cyclosporine A. Whereas immune responses were dampened in these dogs,
-ID activity remained undetectable. In none of the dogs engrafted
with genetically corrected cells was there evidence for clinical
improvement. Our data suggest that, whereas the -ID cDNA may be
transferred and maintained in approximately 5% of hematopoietic
progenitors, the potential of this approach appears limited by the
levels of provirally derived enzyme that are expressed in vivo and by
the host's response to cultured and transduced hematopoietic cells
expressing foreign proteins.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
GENE THERAPIES DELIVERED by hematopoietic
stem cells (HSCs) offer several theoretical advantages over other
methods of drug delivery. These include long-term, regulated, in vivo production of a wide variety of therapeutic agents derived from DNA
permanently integrated into the genomes of cells with life-long capacities for extensive proliferation and multilineage
differentiation.1 Recent progress towards this goal has
been tenuous, with apparent advances generally followed by the
identification of largely unforeseen obstacles. To date there is
limited evidence that genetically modified HSCs can actually be of
therapeutic benefit in human disease or in large animal
models.2-4
Over the last decade, several approaches have been taken to develop
HSCs as efficient vehicles for gene delivery. These include the use of
HSC-enriched cell target populations5 and methods for
selecting gene-modified HSCs.6 One recurring concern is that HSCs, in contrast to their committed progeny, appear to express low levels of the receptor required for successful gene transfer mediated by the retroviral vectors used in most
applications.7 Another problem limiting practical
applications of HSC gene transfer is that the vast majority of HSCs are
likely to be quiescent8 and consequently resistant to
stable gene transfer. Furthermore, the likelihood that any given
gene-modified HSC would be among the small minority that actually
undergo expansion in vivo under steady-state conditions seems
remote.9
We focused on gene transfer to candidate HSCs in in vitro hematopoietic
microenvironments, because it seemed logical that under the correct
culture conditions HSCs may be induced to exit quiescence and enter the
cell cycle. Data from our studies10,11 and from
others12-14 demonstrated that exposure of hematopoietic cells to retroviral vectors in the presence of marrow-derived stromal
cells facilitated reporter gene transfer into a high proportion of
committed progenitors. In a canine model system, autologous marrow
transduced by multiple exposures to retroviral vectors in long-term
marrow cultures (LTMCs) engrafted in the absence of myeloablative
conditioning and gave rise to reporter gene-marked committed progeny
that were maintained at approximately 5% levels for up to 2 years.11 Similar results were more recently obtained from a
human clinical trial of LTMC stem cell gene marking in multiple
myeloma.15 These studies suggested that, although the goal
of achieving large populations of genetically modified hematopoietic cells in vivo remains elusive, the levels of gene transfer achievable using current technology may alleviate disease symptoms in certain deficiency disorders.
To evaluate the therapeutic benefit of low and sustained levels of
genetically modified hematopoietic cells, we attempted gene therapy for
canine  -L-iduronidase (-ID) deficiency. In this autosomal
recessive disorder there is a complete deficiency of -ID due to a
single base substitution in the -ID gene that disrupts intron I
splicing and gives rise to a stop codon.16,17 Dogs
homozygous for the -ID mutation do not have any -ID enzyme as
determined by either -ID activity or
immunoprecipitation.18 The -ID deficiency results in
lysosomal storage of large amounts of the glycosaminoglycans (GAGs)
heparan and dermatan sulfate in many tissues, including the central
nervous system.16,19 -ID-deficient dogs are clinically
similar to human patients with the Hurler/Scheie phenotype of
mucopolysaccharidosis type I (MPS I) and exhibit progressive cardiac
abnormalities, corneal clouding, stunted growth, and degenerative joint
disease, all of which progress to severe states within 2 to 3 years.16,19
We hypothesized that canine -ID deficiency would be an ideal model
system to evaluate the potential clinical benefit of low numbers of
genetically corrected hematopoietic cells for the following reasons:
the disorder is a single gene defect for which the canine cDNA is
cloned; there is a wide range of enzyme levels compatible with a normal
or mild phenotype; matched, related bone marrow transplantation is of
known clinical benefit in affected dogs20; and enzyme
replacement using a human recombinant product is of clinical benefit,
despite the development of complement-activating antibodies.21,22
In this study we infused five homozygous affected -ID-deficient
pups with autologous marrow genetically corrected to express high
levels of functional -ID. Our results demonstrate that long-lived hematopoietic progenitor cells, genetically modified ex vivo, engraft
and are maintained in vivo at levels thought to be clinically relevant.
However, their utility in gene therapy for deficiency states is likely
limited by both the low expression level of the transgene and normal
host immune responses to foreign proteins and cultured transduced
cells. Such responses may be exacerbated when gene-modified autografts
contain large numbers of antigen-presenting cells and their precursors,
such as cultured marrow cells.
| |
MATERIALS AND METHODS |
Dogs and marrow harvests.
The MPS I dogs used in this study were bred and maintained at the
University of Guelph's Central Animal Facility (Guelph, Ontario,
Canada) and were not treated with -ID enzyme before this study. The
animal care and bioethics committees of the Universities of Toronto and
Guelph approved all protocols. Large-scale marrow harvests were
performed on 2- to 12-month-old dogs from either the iliac crest or
proximal humeri and femora under general anesthesia. Marrow, equivalent
in volume to up to 10% of total blood volume, was collected at each
large-scale harvest (range, 100 to 300 mL), whereas approximately 10 mL
marrow was collected at each follow-up time point for analysis.
Light-density marrow cells (<1.075 g/mL) were recovered after Percoll
(Pharmacia, Uppsala, Sweden) density gradient separation
and washing, as previously described.10,11
Retroviral vectors.
Two Moloney-based retroviral vectors were used in this study
(Fig 1). The M48ID vector has extended
viral gag sequences23 upstream of an internal
murine phosphoglycerate kinase-1 promoter,24 which directs
expression of the normal canine -ID cDNA.25 The LCIDSN vector, based on LXSN,26 has the normal canine
-ID cDNA25 expressed from the viral LTR and
neor from the SV40 promoter and was packaged by
PA317.27 The M48ID vector is produced by  CRIP28 and was provided by Drs A. Salvetti and O. Danos
(Paris, France).
View larger version (10K):
[in this window]
[in a new window]
| Fig 1.
Illustration of retroviral vectors. Abbreviations: LTR,
long terminal repeats;  +, packaging signal; gag, gag
sequences; SV, SV40 promoter; neor, neomycin
phosphotransferase; muPGK, murine phosphoglycerate kinase-1 promoter.
|
|
The titers of all producer lines were approximately 1 to 5 × 105 colony-forming units (CFU)/mL determined
on NIH3T3 as previously described.29 To ensure that stocks
were free of replication competent retrovirus (RCR), supernatants from
all producer cell lines were routinely assayed by a sensitive marker
rescue assay.30
Marrow cultures, transductions, and infusions.
Marrow was set up in LTMCs and transduced as described
previously,10,31 with minor modifications. Briefly, 1 × 108 cells were seeded in 150-cm2 tissue
culture flasks (Corning, Corning, NY) in LTMC media
consisting of 10% fetal bovine serum (FBS) and 15% horse serum (HS)
in McCoy's 5A media supplemented as described.29 Control
flasks were initiated and maintained with LTMC media alone and
transduced cultures were initiated in a 50:50 mix of fresh LTMC media
and retroviral supernatant. Retroviral supernatant was prepared by
conditioning LTMC media on confluent retroviral producer cells for 18 to 24 hours at 33°C. Viral supernatants were filtered through a
0.45-µm filter and either used directly or stored at  70°C
until use. All cultures were incubated in a humidified environment at
33°C and 5% CO2 in air for 21 days. LTMCs were
maintained by removal of half of the nonadherent cells and media at
weeks 1 and 2 and replenishment with fresh LTMC media. Twenty-four
hours after feeding, half of the nonadherent cells and media were again
removed and replaced with retroviral supernatant.
The adherent LTMC cells were harvested by enzymatic digestion with
0.25% trypsin and 1 mmol/L EDTA (GIBCO, Grand Island, NY) and washed twice with Hank's buffered saline. Aliquots of cells were
set aside for molecular assays, histology, and marker rescue assay for
RCR. The remaining cells were either cryopreserved until use or infused
directly as previously described.32 Where dogs received
multiple large-scale infusions, time points postinfusion for polymerase
chain reaction (PCR) analysis were calculated from the
first infusion. Tissues at postmortem examination were prepared for
histopathologic analysis.19
In vitro assays of gene transfer.
Proviral marking in hematopoietic progenitors was evaluated by PCR
amplification of proviral specific sequences in individual colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Adherent layer cells were plated in
quadruplicate at a density of 1 × 105 cells/mL in
complete methylcellulose with either recombinant human or murine
cytokines (MethoCult GF M3434 or H4435; Stem Cell Technologies,
Vancouver, British Columbia, Canada) and supplemented with
10% canine phytohemagglutinin (PHA)-stimulated leukocyte conditioned media. The colonies were assessed using standard criteria on days 10 to 12 of culture.33 Up to 100 well-isolated
colonies and 10 methylcellulose only controls were plucked from each
sample. Individual colonies and background methylcellulose controls
were placed into 40 µL of lysis buffer (0.5% NP-40, 0.5% tween-20, and 0.9 mg/mL proteinase-K), incubated at 56°C for 1 to 2 hours, and boiled for 10 minutes to inactivate the proteinase-K. Five microliters of each sample was used per PCR amplification.
Proviral -ID sequences were detected by PCR amplification of a
422-bp amplicon from the -ID cDNA sequence (the amplicon from -ID
genomic DNA is ~1 kb). The -ID sense primer was derived from exon
3 (cID3S; 5'-CAAGGCCTGAGCTACAACTTC-3') and the antisense primer from exon 6 (cID6AS; 5'-GTGCCGTTGTGACAATGCTCC-3').
Proviral -ID PCR was performed with denaturation at 94°C for 3 minutes, followed by 40 cycles of amplification: denaturation at
94°C for 25 seconds, annealing at 60°C for 25 seconds, and
extension at 72°C for 30 seconds. The neor PCR used in
these studies amplifies a 471-bp fragment with a sense primer
N1 (5'-GAACAAGATGGATTGCACGCAG-3') and the
anti-sense primer YJR2
(5'-GTCCAGATCATCCTGATCGACAAG-3'). The quality of DNA was
assessed by PCR amplification of the canine dystrophin gene with the
sense primer (5'-ACAGTCCTCTACTTCTTC CCACCA-3') and the antisense primer (5'-AATTCACAGAGCTTGCCATGC-3'). PCR
reactions were performed in Stratagene Buffer 10 (Stratagene, La Jolla, CA), with 10 pmol of each primer in a 25 µL reaction. The cycling conditions for these PCR reactions start with a denaturation at 94°C for 3 minutes, followed by 42 cycles of amplification:
denaturation at 94°C for 20 seconds, annealing at 61°C for 25 seconds, and extension at 72°C for 30 seconds.
PCR amplification products of colonies were routinely transferred onto
nylon membrane (Hybond N+; Amersham, Arlington Heights, IL), hybridized with an end-labeled radioactive
oligonucleotide specific for each PCR product, and analyzed by
autoradiography. The sequences of the neor and -ID
probes used are as follows: neor,
5'-CGACCTGTCCGGTGCCCTGAATGAACTGG-3'; and -ID,
5'-CCAGGCTGACCGCTATGACC-3'.
Assays for proviral -ID expression.
-ID activity was assessed in duplicate by the 4-methylumbelliferyl
-iduronide (4MUID) fluorimetric assay.21,22 In brief, samples were homogenized in 10 mmol/L phosphate buffer pH 5.8, containing 0.1 mmol/L dithiothreitol and 0.01% triton-X 100, and lysed
by either 4 cycles of freeze-thawing (for assessment of individual CFU)
or sonication (blood, marrow, and LTMC samples). The reaction was
performed in 0.2 mol/L formate, pH 3.5, with 25 µmol/L 4MUID
(Sigma, St Louis, MO) at 37°C for 1 to 24 hours. Serial dilutions
of 4-methylumbelliferone (4MU) were used to calibrate the fluorimeter.
Negative controls included pretreatment samples, tissue blanks, and
reagent controls. Positive controls consisted of matched tissues from
normal dogs. Liberated 4MU was detected fluorimetrically with 365 nm
excitation and 440 emission filters (assay sensitivity ~1% of normal
levels) in either a Turner Diagnostics cuvette reader (Sunnyvale, CA)
for tissue samples or the Fluoroskan II 96 well reader (Labsystems,
Helsinki, Finland) for CFU. One unit of ID activity was determined
to correspond to the release of 1 nmol of 4MUID substrate per
milligram of protein per hour at 37°C. For analysis of -ID
activity from CFU-GM, individual colonies were plucked in 3 to 5 µL
methylcellulose and resuspended in 25 µL buffer. The whole colony was
used for the assay. Background methylcellulose and reagent controls
were included for each sample as negative controls.
Reverse transcriptase-PCR (RT-PCR) was performed to
detect proviral -ID transcripts. RNA was prepared from blood and
marrow leukocytes, treated with RNase-free DNase, and phenol-chloroform extracted. -ID cDNA was synthesized using an antisense primer derived from exon 7 (5'-GGTCCGCCTCGTCGTTGTAA-3') and
Superscript II reverse transcriptase (GIBCO). The mutation in the
-ID gene in MPS I dogs eliminates the splice site in intron 1 and
thus endogenous transcripts from the mutant gene contain intron 1 and are 450 bp longer than transcripts from the normal canine -ID cDNA
present in the provirus. PCR amplification of the cDNA with a forward
primer from exon 1 (primer CA-1 from Menon et al17; 5'-CGCTGCGGCCCCTGCGG CCCTTCT-3') and the reverse primer
cID6AS described above were used for amplification. This reaction
unambiguously distinguishes transcripts derived from the provirus (618 bp) and those from the endogenous mutant gene (1,068 bp). The PCR
reaction and cycling conditions were the same described above. The
quality of the cDNA samples was assessed by PCR amplification of the
exon 3-6 of the -ID gene that provides the same size fragment from both the normal and the MPS I genomic transcripts. Controls without reverse transcriptase enzyme were included for each sample in all
experiments. For RT-PCR amplification of proviral specific transcripts,
primers were chosen that span intron 1.
Immunoassays.
Sera from dogs were tested for the presence of IgG antibodies directed
against -ID, HS, FBS, and bovine serum albumin by enzyme-linked
immunosorbent assay (ELISA). Serum samples were collected
at 1- to 3-month intervals postinfusion. Preinfusion serum was
collected from each dog and used as individual baseline controls. ELISA
was performed as described21; however, wells were blocked
with 0.1% tween-20. For the detection of antibodies against serum or
serum components, wells were coated with 100 µL antigen solution
containing either 0.2 µg recombinant -ID (kindly provided by Dr E. Kakkis, University of California San Francisco, San Francisco, CA) or a
2.5% solution of either bovine serum albumin, HS, or FBS in
phosphate-buffered saline. The secondary antibody used
was alkaline phosphatase-conjugated goat anticanine IgG (Chemicon,
Temecula, CA). The optical density was read on a microtiter plate
reader at 405 nm (Molecular Devices, Sunnyvale, CA). The
titer was reported as the greatest dilution of serum that had an
optical density of greater than twice that of the preimmune serum.
A cellular immune response was assessed by evaluating the proliferation
of peripheral blood mononuclear cells in response to transduced
autologous stroma. Marrow stroma was grown similarly to LTMCs, except
that 4 to 24 hours after culture initiation the nonadherent fraction
was removed and replaced with fresh LTMC media. The stromal cultures
were maintained in LTMC media by twice weekly half volume media
changes. At confluence (10 to 14 days), the stroma was trypsinized and
replated at a 1:3 dilution. Twenty-four hours later, the stroma was
transduced by replacing the media with retroviral supernatant twice
daily for 2 days. Control untransduced stroma was manipulated similarly
to transduced stroma; however, fresh LTMC media was used instead of
retroviral supernatant. Stroma was maintained by 1:3 dilutions once
confluent. At passage 3 to 4, the stromal cells were plated in 100 µL
of Iscove's modified Dulbecco's medium (IMDM) with 15% FBS in
96-well plates. Responder cells were fresh or cryopreserved autologous,
ficoll gradient-separated peripheral blood mononuclear cells and were
resuspended in IMDM with 15% FBS and either 5% canine or 10% murine
leukocyte-conditioned media. One hundred microliters of responder cells
was added at a ratio of 1:10 stimulator to responder cells in
triplicate to each transduced and control stroma sample. The positive
control wells received 2% PHA, whereas blank wells, peripheral blood
cells alone, and stroma alone served as negative controls. Cells were incubated at 37°C for 6 days and during the last 18-hour cells were
pulsed with [3H]-thymidine. Thymidine incorporation was
analyzed on a Wallac 1205 Betaplate counter (Wallac, Gaithersburg, MD)
and reported as the mean cpm (±SD) for triplicate measurements.
| |
RESULTS |
Autografts.
Large volume marrow harvests were performed on five MPS I-affected
dogs. When yields of marrow mononuclear cell were less than 2 × 108 cells/kg, dogs underwent multiple marrow harvests. The
time intervals between collections ranged from 1 to 3 months and in all
cases subsequent harvests were performed after blood cells counts had normalized. Three dogs (M3, M5, and M6) underwent one marrow harvest, whereas dogs M4 and M2 underwent two and three marrow aspirations, respectively. On average, 1.0 × 108 (standard error
[SE]: ±1.6 × 107) mononuclear
cells and 5.4 × 104 (SE: ±4.2 × 103) CFU-GM/kg were obtained from each marrow sample after
density gradient separation and washing.
LTMCs from MPS I dogs were transduced by three exposures to retroviral
vectors bearing the canine -ID cDNA. During 3 weeks of culture,
LTMCs from dogs M3, M4, and M5 were transduced with M48ID (Fig 1).
For the remaining two dogs, LTMCs from the first two marrow harvests
from dog M2 were transduced with M48ID and the third with LCIDSN,
whereas for dog M6, approximately one half of the LTMCs were transduced
with each vector. The cell and progenitor recoveries at the end of the
3-week LTMC and transduction period were 1.5 × 107
cells (SE: ±5.2 × 107) and 9.2 × 103 CFU-GM (SE: ±4.8 × 103) per
kilogram, corresponding to 15% and 17% recovery of cells and CFU-GM.
Gene transfer and -ID expression in transduced LTMCs.
Successful transduction of LTMC cells was confirmed by PCR detection of
proviral DNA in cells from all adherent layers
(Fig 2A and
Table 1). Transduced LTMCs were assayed for
proviral -ID expression. LTMC cells from transduced MPS I cultures
had -ID enzyme activities of between 10 and 198 U (nanomoles per
milligram of protein per hour; Table 1), corresponding to approximately 10 to 200 times normal enzyme levels. There was no activity detected in
untransduced MPS I control LTMC adherent layer cells. All transduced LTMCs were also positive for proviral -ID transcripts by RT-PCR (Fig 3A and Table 1).
View larger version (59K):
[in this window]
[in a new window]
| Fig 2.
Detection of proviral sequences in MPS I
tissues. (A) Proviral -ID and genomic control dystrophin (MD) PCR on
day 21 transduced adherent layer LTMC cells from dogs M2 through M6.
The vectors used for each culture were M-M48ID and L-LCIDSN.
Positive controls were 10% M48ID producer cell line DNA mixed with
90% untransduced MPS I canine DNA and 1% M48ID producer cell line
DNA mixed with 99% untransduced MPS I canine DNA. All samples and
control DNA mixes were positive for dystrophin sequences, and all
transduced LTMCs were positive for proviral -ID. Untransduced LTMC
DNA from dog M6 (UM6) and reagent controls were negative for proviral
sequences. (B) Semiquantitative neor and genomic control
dystrophin PCR on LTMCs transduced with M48ID (L1) and LCIDSN
(L2); postinfusion blood (PB) and marrow (BM) cells from M2 with
0.01%, 0.1%, 1%, and 10% positive control mixed with untransduced
canine DNA; and negative untransduced control (0). (C) PCR
amplification and Southern blot analysis of 10 CFU from M2 at 1 year
postinfusion; dystrophin genomic control (MD), proviral -ID (ID),
and neor (neo) PCR analysis. This sample demonstrates 10 CFU positive for genomic DNA (MD) and 1 of 10 CFU positive for both
-ID and neor sequences (CFU 5). Abbreviations: M, marker
lane; MC, methylcellulose control; RC, reagent control; +C, positive
control DNA.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Evaluation of Gene Transfer and Proviral Gene Expression
Into Canine MPS I LTMC Adherent Layers and Individual CFU-GM
Derived From Transduced LTMCs
|
|
View larger version (34K):
[in this window]
[in a new window]
| Fig 3.
-ID expression evaluated by RT-PCR. (A) LTMC adherent
layers from all dogs and (B) blood (PB) and marrow (BM) leukocytes from
M2 at 1 to 3 months postinfusion. PCR amplification of a 618-bp
proviral specific -ID transcript (proviral transcript) was
performed. PCR amplification of the 422-bp transcript arising from
either the normal or mutant genomic -ID cDNA (endogenous transcript)
was performed to confirm the presence of amplifiable cDNA in these
tissues. A control for each sample was not treated with reverse
transcriptase (RT) to ensure that there was not genomic DNA
contamination of samples. Abbreviations: ULTMC, untransduced LTMC
control from M2; RC, reagent control; +C, positive control DNA.
|
|
To evaluate the levels of gene transfer and expression in the
hematopoietic progenitor subpopulation of LTMCs, individual hematopoietic colonies were assayed. The average percentage of LTMC-derived CFU-GM positive for provirus was 32.8% (range, 20% to
62%; Table 1). Proviral -ID expression was assayed in CFU-GM from
dogs M2 through M5 and on average 15% (range, 8% to 20%) of CFU-GM
from transduced LTMCs had proviral -ID activity. These data indicate
that on average half of the progenitors carrying proviral sequences
expressed the transgene. The in vitro gene transfer efficiencies
obtained in this study were comparable with results obtained in other
canine and human gene transfer studies in our
laboratory.11,15
Infusion of transduced LTMCs.
An average of 1.67 × 107 (range, 9.6 × 106 to 3.1 × 107) LTMC cells per kilogram
were infused into unconditioned autologous recipient dogs that had not
received any type of -ID enzyme therapy
(Fig 4). Dogs were not myeloablated or
otherwise preconditioned. Three dogs (M3, M5, and M6) each received one
infusion. M4 received two infusions of ID-transduced LTMC cells.
After M2 had received three infusions of transduced cells, he was
placed on cyclosporine A immunosuppressive therapy and two more
infusions of 5 × 106 LTMC cells/kg were administered.
Dogs M3 through M6 received immediate posttransplant cyclosporine A and
dog M6 received, in addition, methotrexate (0.2 mg/kg) intravenously on
days 1, 3, 6, and 11 and weekly thereafter.34 The dose of
cyclosporine A used was 10 to 30 mg/kg (cyclosporine A was kindly
provided by Sandoz Canada, Dorval, Quebec, Canada) and adjusted to
maintain a whole blood cyclosporine A level of 400 to 500 ng/mL. LTMC
cells were adminstered intravenously in 50 mL Hank's buffered saline over 15 to 30 minutes. The injections were well tolerated and there
were no late complications.
View larger version (8K):
[in this window]
[in a new window]
| Fig 4.
Summary of LTMC cells infused per kilogram into MPS I
dogs. Multiple infusions in M2 and M4 were separated by 3 to 4 weeks.
Cell doses administered in multiple infusions are indicated by
different shading patterns.
|
|
Gene transfer into in vivo repopulating cells.
To assess the presence of transduced, long-lived, hematopoietic cells
and progenitors, PCR analyses for proviral specific neor
and -ID sequences were performed on samples at up to 36 months postinfusion. Semiquantitative neor PCR analysis was
performed on blood and marrow leukocytes from the two dogs (M2 and M6)
that received the neor containing vector, LCIDSN. The
proportion of neor-positive cells from both dogs was
similar and data from M2 are shown in Fig 2B. The proportion of blood
and marrow leukocytes carrying the proviral genome was between 0.1%
and 1% at time points up to 26 months postinfusion (Fig 2B). The
levels generally decreased to approximately 0.01% at 28 months
postinfusion and were undetectable by 36 months.
The proportion of hematopoietic progenitors carrying the proviral
-ID cDNA was evaluated by PCR amplification of proviral DNA in
marrow-derived hematopoietic colonies (CFU-GM and BFU-E) at time points
after adoptive transfer. A representative CFU PCR analysis comparing
endogenous control of dystrophin PCR, neor PCR, and -ID
PCR from M2 at 12 months postinfusion is shown in Fig 2C. There was
good evidence for the presence of committed progenitors bearing
proviral DNA in all dogs, and all had provirus positive CFU at all time
points tested (at least 1 year postinfusion). Dog M5, for example,
maintained between 2% and 3% provirally marked CFU throughout the
12-month period postinfusion. Other dogs generally had a peak of 5% to
10% positive CFU in the first 6 months postinfusion and then a gradual
decrease to 2% to 5% at time points greater than 1 year
(Table 2 and Fig 5). Dog
M2 was observed the longest and maintained levels of up to 6% provirus
positive CFU between 12 and 24 months and 2% to 5% at up to 24 to 36 months postinfusion. Generally, the level of progenitors carrying
provirus in the MPS I dogs in this study were similar to those observed
in normal dogs used in previous marker gene studies from our
center.11 For negative controls, background methylcellulose
from each plate and colonies from three marrow samples from control,
untreated dogs were harvested, plucked, and analyzed by PCR. A total of 139 colonies from untransduced control dogs and 177 background methylcellulose plucking controls from treated dogs underwent neor and/or -ID PCR analysis and all were
negative.
View this table:
[in this window]
[in a new window]
|
Table 2.
Detection of Proviral-Specific Sequences in
Marrow-Derived Hematopoietic Colonies (CFU-GM and BFU-E) From MPS I
Dogs at Time Points Postinfusion
|
|
View larger version (13K):
[in this window]
[in a new window]
| Fig 5.
Percentage of provirus-positive progenitors in five MPSI
(M2 through M6) dogs observed for 1 to 2 years postinfusion. At each
time point, up to 100 colonies (CFU-GM and/or BFU) and 10 methylcellulose controls were plucked from methylcellulose plates and
subject to -ID or neor PCR.
|
|
Proviral -ID expression in vivo.
Peripheral blood and marrow leukocytes from all five dogs were assayed
for expression by -ID enzyme activity and RT-PCR on multiple
occasions after adoptive transfer. -ID enzyme activity was not
detected in either blood or marrow mononuclear cells from any dog
(sensitivity ~1%). Proviral specific RT-PCR on blood and marrow
mononuclear cells collected during the first 3 months postinfusion from
all dogs were consistently negative (data from dog M2 shown in Fig 3B).
Expression data from two dogs (M2 and M5) are summarized in
Table 3. Proviral -ID
expression was also assessed in hematopoietic progenitors, because this
population of hematopoietic cells had higher levels of provirus (2% to
10%) than the total blood and marrow leukocytes (<1%). The -ID
activity assay was performed on individual CFU-GM from M2 and M5 at
various time points postinfusion. A total of 399 CFU-GM from 1 to 24 months were assayed from M2. One CFU-GM of 155 was positive for -ID
activity at 1 month, whereas 244 CFU-GM from time points up to 24 months were negative. In dog M5, -ID activity was not detected in
any of 193 CFU-GM from 1 to 4 months postinfusion (Table 3). Controls
from these experiments included CFU-GM from normal dogs as positive
controls and reagent and tissue negative controls.
Immune response assays.
Immune responses against provirally marked cells, proviral gene
products, and sera components were evaluated. Specific IgG antibodies
against -ID enzyme were detected by ELISA in sera from dog M2 at all
time points postinfusion. The titer peaked after the third infusion
(~2 months postinfusion) at 1:3,200 and decreased to 1:800 to 1:1,600
after initiation of treatment with cyclosporine A at approximately 4 months postinfusion (Table 4). Humoral
immune responses against FBS, HS, and bovine serum albumin were assayed
after the third infusion and demonstrated IgG titers of 1:3,200 to
1:6,400 (Fig 6). The anti-fetal bovine and
anti-horse sera antibodies observed were maintained in the serum of M2
for at least 1 year after the infusion of transduced cells at titers of
1:1,600 to 1:3,200. The highest anti--ID titer was observed in M2
after the third infusion of gene modified cells. The other dogs with
lower anti--ID antibody titers received only one or two infusions
of gene-modified cells.
View larger version (21K):
[in this window]
[in a new window]
| Fig 6.
Detection of serum IgG specific for -ID (ID), HS, FBS,
and bovine serum albumin (BSA) in dog M2 by ELISA. Titers are shown as
the dilution of serum that gave a corrected OD reading of greater than
two times the dog's preinfusion serum. Analysis of anti-FBS, -HS, and
-BSA antibodies was not evaluated before 3 months. The timing of
infusions and initiation of treatment with cyclosporine A are shown
with arrows.
|
|
Cellular immune responses to autologous transduced cells were evaluated
by peripheral blood mononuclear cell proliferation assays. Two
experiments were performed with blood mononuclear samples from dog M2 2 and 6 weeks after the third infusion of LTMC cells. In these
experiments peripheral blood mononuclear cells were overlaid on
transduced and control autologous stroma and the cell proliferation
under each condition was measured by incorporation of
[3H]-thymidine and reported as mean cpm ± standard
deviation. In the first experiment, fresh peripheral blood mononuclear
cells from 2 weeks after the third infusion were exposed to
LCIDSN-transduced and untransduced control stroma. There was an
approximately threefold increase in [3H]-thymidine
incorporation in LCIDSN-transduced stroma over untransduced controls
at blood mononuclear cells to stroma ratios of 10:1 (7,020 ± 719 cpm LCIDSN transduced stroma v 1,850 ± 383.1 cpm untransduced stroma; P < .005;
Fig 7A). In the second experiment, three
separate stimulatory autologous stromal cells were used: untransduced, transduced with LNc11 (a neor only containing vector), and
M48ID (the vector containing only -ID). Cryopreserved blood
mononuclear cells from 6 weeks after the third infusion of LTMC cells
were used as responders. There was approximately twofold higher
proliferation when blood mononuclear cells were stimulated with
M48ID-transduced autologous stroma versus control untransduced
stroma (5,367 ± 1792 cpm M48ID-transduced stroma v
3,053 ± 812 cpm untransduced stroma; P < .05)
or versus LNc11-transduced stroma (5,367 ± 1,792 cpm
M48ID-transduced stroma v 2,635 ± 487 cpm
LNc11-transduced stroma; P < .05; Fig 7B). There was no
significant difference between stimulation with LNc11-transduced stroma
or untransduced stroma (P = .167). The results from
these two experiments are consistent with a cellular immune response to
-ID but not to neor-transduced cells. The higher rate of
cell proliferation in the second experiment than the first may be due
to a dampening of the immune response with time.
View larger version (32K):
[in this window]
[in a new window]
| Fig 7.
Peripheral blood mononuclear cell (MNC) proliferative
response to autologous LCIDSN-transduced stroma in dog M2. Blood
MNCs from dog M2, which received infusions of both LCIDSN and
M48ID transduced LTMC cells, were cocultured in vitro with 1.5 × 103 autologous LCIDSN transduced ( ) and untransduced
( ) stroma. MNC proliferation is measured by the incorporation of
[3H]-thymidine and reported as the mean counts per minute
(cpm) of triplicate wells with standard errors shown. The times after
third infusion were (A) 2 weeks and (B) 6 weeks. *P < .005 and  P < .05.
|
|
The demonstration of immune responses against -ID, the serum
components of the LTMC media, and autologous cells expressing -ID in
dog M2 after multiple infusions led to the treatment of dogs M3 through
M6 with fewer infusions of transduced cells and immunosuppressive
therapy to abrogate immune responses against -ID and transduced
cells. Three dogs received standard cyclosporine A doses during the
immediate posttransplant period. As expected, the titers of
-ID-specific IgG were lower than those for M2, were at 1:100 in M3
and M5 receiving one infusion, and peaked at 1:400 in M4 after a second
infusion (Table 4). The humoral immune response was
further reduced to background levels in dog M6 treated with one
infusion of cells and both cyclosporine A and methotrexate, because
titers were less than 1:100.
Pathology.
Three MPS I dogs (M3, M5, and M6) were euthanized at 12 to 24 months
postadoptive transfer due to worsening clinical status and increased
morbidity, whereas dog M2 died naturally from disease-related causes at
42 months postinfusion. Pathologic examination of all euthanized
affected dogs was consistent with normal progression of MPS I disease.
Vacuolated cells were prominent in the connective tissues from skin,
gastrointestinal tract, liver, pancreas, heart and arteries,
respiratory system, reproductive system, and urinary and hemolymphatic
systems. Dog M3 is alive at 28 months postinfusion, with advanced but
stable disease.
| |
DISCUSSION |
Canine -ID deficiency was first described in 1982 as a new mutation
in three Plott hound dogs from Tennessee.16 Through thoughtful breeding programs, colonies were established and arising affected dogs have been the subjects of several important experiments designed to learn more about the comparable human condition, Hurler syndrome or MPS I. Allogeneic bone marrow transplantation in
5-month-old affected pups was shown to result in increased -ID
levels in cerebral cortex, liver, and cerebrospinal fluid and reduced
GAG storage in liver, neurons, glial cells, and blood
vessels.20 In transplanted pups, urinary GAG excretion
decreased to normal levels by 5 months posttransplant. Enzyme
replacement studies have also been undertaken in affected dogs using
recombinant human -L-iduronidase.21,22 Intravenous
administration of purified enzyme to three affected pups for up to 13 months21,22 resulted in normalization of lysosomal storage
in liver, spleen, and kidney glomeruli. There was no improvement in
brain tissue GAG storage, and all dogs developed complement-activating
antibodies against the human protein.
The promising results of allogeneic bone marrow
transplantation20 and evidence for clinical improvement in
recombinant enzyme infusion studies, despite the presence of an immune
response,21,22 suggested that -ID-deficient dogs might
be ideal for developing and evaluating therapies delivered by
genetically corrected HSCs. The need for such model systems was
heightened by the generally poor results obtained in several large
animal and human studies of HSC gene transfer. Studies in
cats,35 dogs,10,36 nonhuman primates,5,37 humans,2,38,39 and surrogate
human systems40,41 suggested that only a small proportion
of in vivo repopulating HSCs are genetically modified using methods
that gave high levels of HSC gene transfer in murine systems.
Furthermore, there still is no clear evidence for a therapeutic benefit
of genetically modified HSCs in any large animal or human
system.3,42 We reasoned that the demonstration of
significant disease amelioration in affected -ID-deficient dogs
receiving genetically corrected HSCs would represent a much-needed
proof of principle in the arena of HSC gene therapy.
We performed large volume marrow harvests from five affected
-ID-deficient dogs and exposed hematopoietic cells with known long-term in vivo repopulating potential to two retroviral vectors bearing the normal canine -ID cDNA. One vector, LCIDSN, expressed -ID from the LTR and neor from the SV40
promoter/enhancer. In the M48ID vector, -ID was expressed from
the murine PGK promoter. Both vectors were evaluated by us and
independently by others and shown to be excellent vectors as determined
by in vitro assays of transduction.43 Adoptive transfers of
transduced LTMCs to autologous recipients were performed under
conditions shown in prior studies to give rise to genetically modified
hematopoietic cells with capacities to generate committed progenitors
in vivo for up to 3 years and at levels comprising approximately 5% of
all committed progenitors.11 Our particular approach to
gene transfer has relied heavily on the use of stromal-based culture
systems, because data from several sources suggest that such in vitro
microenvironments may facilitate both maintenance of and gene transfer
into HSCs.10-14,44
There was good evidence that a high proportion of LTMC-derived
committed progenitors carried and expressed the proviral -ID, as
well as the bulk LTMC cells that expressed up to 200 times the normal
-ID enzyme levels in vitro. Monitoring of engraftment for up to 2 years by PCR amplification of proviral sequences demonstrated that up
to 6% of hematopoietic progenitors and 0.01% to 1% of blood and
marrow leukocytes of affected dogs were gene marked. These data confirm
prior results from our center.11 The higher level of
marking in hematopoietic progenitors than differentiated cells observed
in these dogs has also been seen in both our studies of LTMC gene
transfer studies in human patients on a stem cell gene marking
trial15 and in studies by others.2,45 The
reason for this phenomenon is unknown but possibly involves an
inability of transduced progenitors to undergo natural processes of
proliferation and differentiation or immune responses to differentiated
hematopoietic cells presenting foreign antigens.
Despite the documentation of engraftment of up to 1% of blood and
marrow leukocytes and 10% of progenitors carrying the provirus, we
observed no -ID activity in blood or marrow leukocytes. At the time
of infusion, LTMC cells expressed up to 200 times the normal level of
-ID protein and an average of 15% of individual hematopoietic
colonies had proviral -ID enzyme activity. After infusion, -ID
activity was not detected in blood or marrow leukocytes and only 1 of a
total of 592 CFU-GM from dogs M2 and M5 was found to be expressing
-ID, suggesting that proviral gene expression had been silenced in
our vectors. Silencing of Moloney murine leukemia virus
(MMLV)-based promoters, such as that used in these vectors, has been described in other in vitro and in vivo
studies.46-48 In our own previous canine gene transfer
studies with the LN vector that has a similar vector backbone to these
studies, expression of neor was maintained in approximately
5% of CFU-GM for up to 2 years postinfusion. The lack of -ID
proviral expression in MPS I dogs may be due to the presence of two
genes in the LCIDSN vector, a phenomenon that has been associated
with decreased expression of both genes.46 Alternatively
silenced proviral -ID expression in MPS I dogs, but not
neor in normal dogs, might be the result of the anti--ID
immune response noted in MPS I dogs. Inflammatory cytokines such as
tumor necrosis factor- (TNF-) and interferon- (IFN-)
induced by immune responses have been shown to inhibit expression of
MMLV-based promoters.49,50
We also investigated the role of humoral and cellular immune responses
against -ID, LTMC media components, and -ID-transduced cells.
Strong immune responses to -ID enzyme, HS, and FBS components and
-ID-transduced autologous cells were detected in dog M2 after three
infusions of transduced cells. All subsequently infused dogs (M3
through M6) were treated with posttransplant immunosuppression and
received fewer infusions in an attempt to abrogate the immune responses. The humoral anti--ID and antiserum immune responses in
these dogs, although somewhat lower than those of M2, were still present.
Humoral immune responses have been detected against a variety of
nontherapeutic antigens presented by genetically modified cells such as
adenoviral vector proteins51 or serum components of culture
media,52 whereas cellular responses have been detected against marker genes, such as a thymidine kinase-neor
fusion protein.53 However, few gene transfer studies have
used animal models of human disease to evaluate gene therapy
strategies. This study in dogs with -ID deficiency demonstrated
significant humoral immune responses against the normal canine -ID
and LTMC culture media serum components. As expected, the strongest
immune responses in our study were detected after multiple infusions of
cells in the absence of immunosuppressive therapy. To our knowledge, this is the first demonstration of both humoral and cellular immune responses against a potentially therapeutic, normal canine enzyme in
dogs deficient for this protein. Both the presence of cellular immune
responses and lack of detectable -ID expression in vivo may indicate
that only cells that have silenced proviral expression survived.
However, further investigation is needed to confirm this.
Two mechanisms may account for the lack of sustained enzyme production:
silencing of proviral gene expression and immune responses against
-ID produced in vivo and/or cells expressing proviral antigens. In ongoing studies, transduced LTMC grafts have been infused
into preimmune fetal MPS I recipients to evaluate the therapeutic
potential of HSC gene transfer for -ID-deficient dogs in the
absence of immune responses.54 Retroviral vectors that have
been modified to optimize long-term transgene expression, such as the
MND,48 MFG,55 and MSCV56 vectors,
will be evaluated in future studies. These and other approaches may
enable a definitive evaluation of the therapeutic potential of
genetically corrected HSCs in the canine -ID deficiency model system.
| |
ACKNOWLEDGMENT |
The authors express our appreciation to Dr Margaret Hough, Yongjun
Zhao, Xiaochen Lu, and the staff of S.D. Laboratories Ltd (Toronto, Ontario, Canada) for their assistance and cooperation. We are
grateful to Drs Anna Salvetti and Olivier Danos for providing us with
retroviral producer lines and to Dr Emil D. Kakkis for providing the
canine -ID cDNA and purified -ID enzyme and for his generous
assistance with the 4MUID and ELISA assays.
| |
FOOTNOTES |
Submitted March 5, 1998; accepted November 10, 1998.
Supported by grants from the Medical Research Council of Canada.
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.
Address reprint requests to Ian D. Dubé, PhD, Department of
Laboratory Medicine, Sunnybrook Health Science Center, 2075 Bayview Ave
(Room E346), Toronto, Ontario, Canada M4N 3M5; e-mail:
ian.dube{at}utoronto.ca.
| |
REFERENCES |
1.
Karlsson S:
Treatment of genetic defects in hematopoietic cell function by gene transfer.
Blood
78:2481, 1991[Free Full Text]
2.
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
4:775, 1998[Medline]
[Order article via Infotrieve]
3.
Verma IM, Somia N:
Gene therapy Promises, problems and prospects.
Nature
389:239, 1997[Medline]
[Order article via Infotrieve]
4.
Malech HL, Maples PB, Whiting-Theobald N, Linton GF, Sekhsaria S, Vowells SJ, Li F, Miller JA, DeCarlo E, Holland SM, Leitman SF, Carter CS, Butz RE, Read EJ, Fleisher TA, Schneiderman RD, Van Epps DE, Spratt SK, Maack CA, Rokovich JA, Cohen LK, Gallin JI:
Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease.
Proc Natl Acad Sci USA
94:12133, 1997[Abstract/Free Full Text]
5.
Bodine DM, Moritz T, Donahue RE, Luskey BD, Kessler SW, Martin DI, Orkin SH, Nienhuis AW, Williams DA:
Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells.
Blood
82:1975, 1993[Abstract/Free Full Text]
6.
Conneally E, Bardy P, Eaves CJ, Thomas T, Chappel S, Shpall EJ, Humphries RK:
Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer.
Blood
87:456, 1996[Abstract/Free Full Text]
7.
Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline A, Bodine DM:
The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.
Proc Natl Acad Sci USA
93:11097, 1996[Abstract/Free Full Text]
8.
Hodgson GS, Bradley TR:
Properties of haematopoietic stem cells surviving 5-fluorouracil treatment: Evidence for a pre-CFU-S cell?
Nature
281:381, 1979[Medline]
[Order article via Infotrieve]
9.
Abkowitz JL, Catlin SN, Guttorp P:
Evidence that hematopoiesis may be a stochastic process in vivo.
Nat Med
2:190, 1996[Medline]
[Order article via Infotrieve]
10.
Carter RF, Abrams-Ogg AC, Dick JE, Kruth SA, Valli VE, Kamel-Reid S, Dubé ID:
Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors.
Blood
79:356, 1992[Abstract/Free Full Text]
11.
Bienzle D, Abrams-Ogg AC, Kruth SA, Ackland-Snow J, Carter RF, Dick JE, Jacobs RM, Kamel-Reid S, Dubé ID:
Gene transfer into hematopoietic stem cells: Long-term maintenance of in vitro activated progenitors without marrow ablation.
Proc Natl Acad Sci USA
91:350, 1994[Abstract/Free Full Text]
12.
Moore KA, Deisseroth AB, Reading CL, Williams DE, Belmont JW:
Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells.
Blood
79:1393, 1992[Abstract/Free Full Text]
13.
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 (LTCICs) from the bone marrow of a patient with Gaucher disease.
Gene Ther
2:512, 1995[Medline]
[Order article via Infotrieve]
14.
Xu LC, Kluepfel-Stahl S, Blanco M, Schiffmann R, Dunbar C, Karlsson S:
Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients.
Blood
86:141, 1995[Abstract/Free Full Text]
15. Stewart AK, Sutherland DR, Nanji S, Zhao Y, Ruedy C, Lutzko C,
Nayar R, Peck B, McGarrity G, Tisdale J, Dubé ID: Gene marked
long term culture cells contribute at low levels to hematopoiesis after
autologous transplant. (submitted)
16.
Shull RM, Munger RJ, Spellacy E, Hall CW, Constantopoulos G, Neufeld EF:
Canine alpha-L-iduronidase deficiency. A model of mucopolysaccharidosis I.
Am J Pathol
109:244, 1982[Medline]
[Order article via Infotrieve]
17.
Menon KP, Tieu PT, Neufeld EF:
Architecture of the canine IDUA gene and mutation underlying canine mucopolysaccharidosis I.
Genomics
14:763, 1992[Medline]
[Order article via Infotrieve]
18.
Shull RM, Lu X, McEntee MF, Bright RM, Pepper KA, Kohn DB:
Myoblast gene therapy in canine mucopolysaccharidosis. I: Abrogation by an immune response to alpha-L-iduronidase.
Hum Gen Ther
7:1595, 1996[Medline]
[Order article via Infotrieve]
19.
Shull RM, Helman RG, Spellacy E, Constantopoulos G, Munger RJ, Neufeld EF:
Morphologic and biochemical studies of canine mucopolysaccharidosis I.
Am J Pathol
114:487, 1984[Abstract]
20.
Shull RM, Hastings NE, Selcer RR, Jones JB, Smith JR, Cullen WC, Constantopoulos G:
Bone marrow transplantation in canine mucopolysaccharidosis I. Effects within the central nervous system.
J Clin Invest
79:435, 1987
21.
Shull RM, Kakkis ED, McEntee MF, Kania SA, Jonas AJ, Neufeld EF:
Enzyme replacement in a canine model of Hurler syndrome.
Proc Natl Acad Sci USA
91:12937, 1994[Abstract/Free Full Text]
22.
Kakkis ED, McEntee MF, Schmidtchen A, Neufeld EF, Ward DA, Gompf RE, Kania S, Bedolla C, Chien SL, Shull RM:
Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysaccharidosis I.
Biochem Mol Med
58:156, 1996[Medline]
[Order article via Infotrieve]
23.
Guild BC, Finer MH, Housman DE, Mulligan RC:
Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo.
J Virol
62:3795, 1988[Abstract/Free Full Text]
24.
Adra CN, Boer PH, McBurney MW:
Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter.
Gene
60:65, 1987[Medline]
[Order article via Infotrieve]
25.
Stoltzfus LJ, Sosa-Pineda B, Moskowitz SM, Menon KP, Dlott B, Hooper L, Teplow DB, Shull RM, Neufeld EF:
Cloning and characterization of cDNA encoding canine alpha-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog.
J Biol Chem
267:6570, 1992[Abstract/Free Full Text]
26.
Miller AD, Rosman GJ:
Improved retroviral vectors for gene transfer and expression.
Biotechniques
7:980, 1989[Medline]
[Order article via Infotrieve]
27.
Miller AD, Buttimore C:
Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.
Mol Cell Biol
6:2895, 1986[Abstract/Free Full Text]
28.
Danos O, Mulligan RC:
Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.
Proc Natl Acad Sci USA
85:6460, 1988[Abstract/Free Full Text]
29.
Dubé ID, Kruth S, Abrams-Ogg A, Kamel-Reid S, Lutzko C, Nanji S, Ruedy C, Singaraja R, Wild A, Krygsman P, Chu P, Messner H, Reddy V, McGarrity G, Stewart AK:
Preclinical assessment of human hematopoietic progenitor cell transduction in long-term marrow cultures.
Hum Gen Ther
7:2089, 1996[Medline]
[Order article via Infotrieve]
30.
Allan DS, De Koven A, Wild A, Kamel-Reid S, Dubé ID:
Endogenous murine leukemia virus DNA sequences in murine cell lines: Implications for gene therapy safety testing by PCR.
Leuk Lymphoma
23:375, 1996[Medline]
[Order article via Infotrieve]
31.
Carter RF, Kruth SA, Valli VE, Dubé ID:
Long-term culture of canine marrow: Cytogenetic evaluation of purging of lymphoma and leukemia.
Exp Hematol
18:995, 1990[Medline]
[Order article via Infotrieve]
32.
Abrams-Ogg AC, Kruth SA, Carter RF, Valli VE, Kamel-Reid S, Dubé ID:
Preparation and transfusion of canine platelet concentrates.
Am J Vet Res
54:635, 1993[Medline]
[Order article via Infotrieve]
33.
Coulombel L, Eaves AC, Eaves CJ:
Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer.
Blood
62:291, 1983[Abstract/Free Full Text]
34.
Nash RA, Pepe MS, Storb R, Longton G, Pettinger M, Anasetti C, Appelbaum FR, Bowden RA, Deeg HJ, Doney K, Martin PJ, Sullivan KM, Sanders J, Witherspoon RP:
Acute graft-versus-host disease: analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate.
Blood
80:1838, 1992[Abstract/Free Full Text]
35.
Lothrop CD J, al-Lebban ZS, Niemeyer GP, Jones JB, Peterson MG, Smith JR, Baker HJ, Morgan RA, Eglitis MA, Anderson WF:
Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow.
Blood
78:237, 1991[Abstract/Free Full Text]
36.
Kiem HP, Darovsky B, Von Kalle C, Goehle S, Graham T, Miller AD, Storb R, Schuening FG:
Long-term persistence of canine hematopoietic cells genetically marked by retrovirus vectors.
Hum Gen Ther
7:89, 1995[Medline]
[Order article via Infotrieve]
37.
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
93:11871, 1996[Abstract/Free Full Text]
38.
Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC, Buschle M, Krance RA, Santana VM, Anderson WF, Ihle JN:
Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.
Lancet
342:1134, 1993[Medline]
[Order article via Infotrieve]
39.
Dunbar CE, Cottler-Fox M, O'Shaughnessy JA, Doren S, Carter C, Berenson R, Brown S, Moen RC, Greenblatt J, Stewart FM, Leitman SF, Wilson WH, Cowan K, Young NS, Neinhuis A:
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood
85:3048, 1995[Abstract/Free Full Text]
40.
Nolta JA, Dao MA, Wells S, Smogorzewska EM, Kohn DB:
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.
Proc Natl Acad Sci USA
93:2414, 1996[Abstract/Free Full Text]
41.
Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE:
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy.
Nat Med
2:1329, 1996[Medline]
[Order article via Infotrieve]
42.
Ross G, Erickson R, Knorr D, Motulsky AG, Parkman R, Samulski J, Straus SE, Smith BR:
Gene therapy in the United States: A five-year status report.
Hum Gen Ther
7:1781, 1996[Medline]
[Order article via Infotrieve]
43.
Shull RM, Lu X, Dubé I, Lutzko C, Kruth S, Abrams-Ogg A, Kiem HP, Goehle S, Schuening F, Millan C, Carter R:
Humoral immune response limits gene therapy in canine MPS I.
Blood
88:377, 1996[Free Full Text]
44.
Barnett MJ, Eaves CJ, Phillips GL, Gascoyne RD, Hogge DE, Horsman DE, Humphries RK, Klingemann HG, Lansdorp PM, Nantel SH, Reece DE, Shepherd JD, Spinelli JJ, Sutherland HJ, Eaves AJ:
Autografting with cultured marrow in chronic myeloid leukemia: Results of a pilot study.
Blood
84:724, 1994[Abstract/Free Full Text]
45.
Brenner MK, Rill DR, Moen RC, Krance RA, Mirro J Jr, Anderson WF, Ihle JN:
Gene-marking to trace origin of relapse after autologous bone-marrow transplantation.
Lancet
341:85, 1993[Medline]
[Order article via Infotrieve]
46.
Bowtell DD, Cory S, Johnson GR, Gonda TJ:
Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes.
J Virol
62:2464, 1988[Abstract/Free Full Text]
47.
Challita PM, Kohn DB:
Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
Proc Natl Acad Sci USA
91:2567, 1994[Abstract/Free Full Text]
48.
Robbins PB, Skelton DC, Yu XJ, Halene S, Leonard EH, Kohn DB:
Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells.
Proc Natl Acad Sci USA
95:10182, 1998[Abstract/Free Full Text]
49.
Harms JS, Splitter GA:
Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter.
Hum Gene Ther
6:1291, 1995[Medline]
[Order article via Infotrieve]
50.
Qin L, Ding Y, Pahud DR, Chang E, Imperiale MJ, Bromberg JS:
Promoter attenuation in gene therapy: Interferon- and tumor necrosis factor- inhibit transgene expression.
Hum Gen Ther
8:2019, 1997[Medline]
[Order article via Infotrieve]
51.
Fang B, Eisensmith RC, Wang H, Kay MA, Cross RE, Landen CN, Gordon G, Bellinger DA, Read MS, Hu PC, Brinkhous KM, Woo SLC:
Gene therapy for hemophilia B: Host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.
Hum Gene Ther
6:1039, 1995[Medline]
[Order article via Infotrieve]
52.
Muul L:
Production and evaluation of retroviral vectors for clinical gene therapy. 2nd Conference on Stem Cell Gene Therapy: Biology and Technology. Orcas Island, WA, 1998.
53.
Riddell SR, Elliott M, Lewinsohn DA, Gilbert MJ, Wilson L, Manley SA, Lupton SD, Overell RW, Reynolds TC, Corey L, Greenberg PD:
T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients.
Nat Med
2:216, 1996[Medline]
[Order article via Infotrieve]
54. Lutzko C, Omori F, Abrams-Ogg ACG, Shull RM, Liheng L, Lau K,
Ruedy C, Nanji S, Gartley C, Dobson H, Foster R, Kruth S, Dubé
ID: Gene therapy for canine -L-iduronidase deficiency: In utero
adoptive transfer of genetically corrected hematopoietic progenitors
results in engraftment but not amelioration of disease.
(submitted)
55.
Riviere I, Brose K, Mulligan RC:
Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.
Proc Natl Acad Sci USA
92:6733, 1995[Abstract/Free Full Text]
56.
Hawley RG, Lieu FH, Fong AZ, Hawley TS:
Versatile retroviral vectors for potential use in gene therapy.
Gen Ther
1:136, 1994[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. Traversari, S. Marktel, Z. Magnani, P. Mangia, V. Russo, F. Ciceri, C. Bonini, and C. Bordignon
The potential immunogenicity of the TK suicide gene does not prevent full clinical benefit associated with the use of TK-transduced donor lymphocytes in HSCT for hematologic malignancies
Blood,
June 1, 2007;
109(11):
4708 - 4715.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Passini, J. Bu, J. A. Fidler, R. J. Ziegler, J. W. Foley, J. C. Dodge, W. W. Yang, J. Clarke, T. V. Taksir, D. A. Griffiths, et al.
Combination brain and systemic injections of AAV provide maximal functional and survival benefits in the Niemann-Pick mouse
PNAS,
May 29, 2007;
104(22):
9505 - 9510.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Berger, C. A. Blau, M.-L. Huang, J. D. Iuliucci, D. C. Dalgarno, J. Gaschet, S. Heimfeld, T. Clackson, and S. R. Riddell
Pharmacologically regulated Fas-mediated death of adoptively transferred T cells in a nonhuman primate model
Blood,
February 15, 2004;
103(4):
1261 - 1269.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Andersson, B. M. W. Illigens, K. W. Johnson, D. Calderhead, C. LeGuern, G. Benichou, M. E. White-Scharf, and J. D. Down
Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice
Blood,
June 1, 2003;
101(11):
4305 - 4312.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. P. Ponder, J. R. Melniczek, L. Xu, M. A. Weil, T. M. O'Malley, P. A. O'Donnell, V. W. Knox, G. D. Aguirre, H. Mazrier, N. M. Ellinwood, et al.
From the Cover: Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs
PNAS,
October 1, 2002;
99(20):
13102 - 13107.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. D. Brown and D. Lillicrap
Dangerous liaisons: the role of "danger" signals in the immune response to gene therapy
Blood,
July 30, 2002;
100(4):
1133 - 1140.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. D. Tran, C. D. Porada, G. Almeida-Porada, H. A. Glimp, W. F. Anderson, and E. D. Zanjani
Induction of stable prenatal tolerance to {beta}-galactosidase by in utero gene transfer into preimmune sheep fetuses
Blood,
June 1, 2001;
97(11):
3417 - 3423.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Rosenzweig, M. Connole, R. Glickman, S.-P. S. Yue, B. Noren, M. DeMaria, and R. P. Johnson
Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34+ hematopoietic cells
Blood,
April 1, 2001;
97(7):
1951 - 1959.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Berger, M.-L. Huang, M. Gough, P. D. Greenberg, S. R. Riddell, and H.-P. Kiem
Nonmyeloablative Immunosuppressive Regimen Prolongs In Vivo Persistence of Gene-Modified Autologous T Cells in a Nonhuman Primate Model
J. Virol.,
January 15, 2001;
75(2):
799 - 808.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Hanazono, K. Terao, and K. Ozawa
Gene Transfer into Nonhuman Primate Hematopoietic Stem Cells: Implications for Gene Therapy
Stem Cells,
January 1, 2001;
19(1):
12 - 23.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
C. Berger, S. Xuereb, D. C. Johnson, K. S. Watanabe, H.-P. Kiem, P. D. Greenberg, and S. R. Riddell
Expression of Herpes Simplex Virus ICP47 and Human Cytomegalovirus US11 Prevents Recognition of Transgene Products by CD8+ Cytotoxic T Lymphocytes
J. Virol.,
May 15, 2000;
74(10):
4465 - 4473.
[Abstract]
[Full Text]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION