|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
GENE THERAPY
From the IST-Viral and Molecular Oncology
Section, the Department of Oncology and Surgical Sciences, and the
Department of Pediatrics, University of Padua, Italy; and the Institute
of Virology, University of Veterinary Sciences, Vienna, Austria.
Locus control region (LCR) sequences are involved in the
establishment of open chromosomal domains. To evaluate the possibility of exploiting the human CD2 LCR to regulate gene expression by Moloney
murine leukemia virus (Mo-MLV)-based retroviral vectors in T cells, it
was included in vectors carrying the enhanced green fluorescence
protein (EGFP) reporter gene; then transduction in vitro of lymphoid
and nonlymphoid cell lines was performed. Deletion of the viral
enhancer in the Mo-MLV long terminal repeat was necessary to detect LCR
activity in the context of these retroviral vectors. It was found that
a full-length (2.1 kb), but not a truncated (1.0 kb), CD2 LCR retained
the ability to modulate reporter gene expression by Mo-MLV-derived
retroviral vectors, leading to a homogeneous, unimodal pattern of EGFP
expression that remained unmodified in culture over time, specifically
in T-cell lines; on the other hand, viral titer was strongly reduced
compared with vectors not carrying the LCR. Lentiviral vectors
containing the CD2 LCR could be generated at higher titers and were
used to analyze its effects on gene expression in primary T cells.
Subcutaneous implantation of genetically modified cells in
immunodeficient mice showed that retroviral vectors carrying the CD2
LCR conferred an advantage in terms of transgene expression in vivo,
compared with the parental vector, by preventing the down-modulation of EGFP expression. These findings suggest a potential application of this
LCR to increase gene expression by retroviral and lentiviral vectors in
T lymphocytes.
(Blood. 2001;98:3607-3617) One of the major obstacles to gene transfer in
mammalian cells is the gap between gene expression of cellular genes
from their genomic loci, which usually occurs at adequate levels, and
the relatively poor expression levels obtained when the same gene is
expressed from viral vectors, in many cases from heterologous viral
promoters. This commonly observed phenomenon clearly constitutes a
significant limitation both of gene therapy strategies aimed at
treating genetic diseases by transduction of hematopoietic stem cells
and of protocols of gene transfer into differentiated primary cells,
such as lymphocytes and fibroblasts.1 Differences in gene
expression from genomic loci and viral vectors clearly involve multiple
levels of complexity, including lack of introns in the transgene,
differences between viral and cellular promoters, and absence of some
regulatory sequences in viral vectors In some cases, the immune system also may contribute significantly to
limiting transgene expression in vivo, specifically when the transgene
itself or the viral vector used (ie, adenoviral vectors) is highly
immunogenic. Furthermore, the pattern of expression of virally carried
therapeutic genes and normal cellular genes is strikingly different:
retrovector-encoded genes are expressed heterogeneously in the
transduced cells because of their random integration into the host
genome, whereas most cellular genes are expressed at homogeneous levels
in a defined cell subset. Given these limitations of current gene
transfer methods, LCR sequences have recently attracted attention
because they might improve the level of gene expression by retroviral
vectors (reviewed in2-4). Many studies focused on the
To address this issue, we studied an LCR from the human
CD2 gene. This 2.1-kb sequence has been largely
studied in transgenic mice, in which it leads to high-level,
position-independent gene expression in the T-cell
lineage.8-10 Our findings suggested that CD2
LCR could be useful for improving gene expression by retroviral and
lentiviral vectors in T cells in vitro and in vivo.
Plasmids
PCR was performed in 50 µL standard buffer containing 0.2 µM each
primer and 1 U Taq polymerase (PerkinElmer, Foster City, CA) under the
following conditions: denaturation for 1 minute at 94°C, annealing
for 30 seconds at 65°C, extension for 2 minutes at 72°C, for 30 cycles, with 5 ng plasmid DNA as template. PCR fragments were purified
and cloned in the pTA2 vector (Invitrogen, Groningen, The Netherlands).
Subsequently, PCR products were excised from this vector by digestion
with NotI or XbaI and were sticky-end cloned by
standard procedures in the corresponding sites of a retroviral vector
termed LESN, which was used as the basic vector, or derivatives of it.
The LESN vector (Figure 1B) is a derivative of the LXSN retroviral
vector13 and carries the gene for enhanced green
fluorescence protein (EGFP) driven by the Mo-MLV long terminal repeat
(LTR) and a neomycin resistance (neoR) cassette (Figure 1B). To
investigate LCR function in the absence of the viral enhancer, we
generated a modified LESN retroviral vector carrying a deletion in the
U3 region of the LTR between bases 2976 and 3186 of the parental LXSN
genome13 and termed it LESN The Mo-MLV Gag-Pol expression construct
gag-polgpt harbors the Mo-MLV
gag and pol genes under the control of the Mo-MLV
LTR and a SV40 polyadenylation signal, but it lacks Cell culture and transfections
Transduction of cells with retroviral vectors
In experiments with lymphoid cells, because of the low titer of the LCR-carrying vectors, all the relevant supernatants were concentrated 100-fold by ultracentrifugation as described.20 Transduction was performed by incubating 1 mL retroviral vector-containing supernatant with 2 × 105 target cells for 6 to 9 hours at 37°C in the presence of protamine sulfate (8 µg/mL). Lymphoid cells were then grown for an additional 4 weeks in G418-containing medium (500 µg/mL) before assessment of EGFP expression. Proviral DNA analysis Genomic DNA was obtained from vector-transduced Molt-3 and NIH-3T3 cells using the EasyDNA kit (Invitrogen). For Southern blot analysis, 20 µg DNA was digested to completion with either KpnI or XhoI/HindIII (New England Biolabs, Beverly, MA), electrophoresed on 0.8% agarose gels, transferred to nylon membranes (Amersham-Pharmacia Biotech, Little Chalfont, United Kingdom) by Southern blotting as described,21 and hybridized to a 32P-labeled 1.32-kb HindIII/SmaI DNA probe from plasmid pSV2-neo. To determine provirus copy number in the transduced cells, the blot was stripped and rehybridized with a loading control probe (CXCR4).22 After washes under high-stringency conditions, filters were autoradiographed for 48 to 96 hours at 70°C. Band
intensities were quantified with an InstantImager (Packard, Meriden,
CT) and were used to calculate the relative number of provirus copies per genome in each sample.
Lentiviral vector-mediated gene transfer in primary T cells All lentiviral vectors were derived from ViG BH, which is an
simian immunodeficiency virus (SIV)-based lentiviral vector
carrying an internal EGFP gene driven by an MLV LTR (details
of their construction are available on request).23
Vector-containing supernatants were generated by a 3-plasmid vector
packaging system based on transient transfections of 293T cells with 6 µg Hgp, a synthetic HIV gag-pol expression plasmid,24 6 µg ViGBH or its derivatives, and 0.1 µg VSV-G expression
construct, as reported for Mo-MLV-based retroviral vectors. NIH-3T3
cells were used in preliminary experiments for titration of the
vectors. Peripheral blood lymphocytes from healthy donors were
centrifuged on Ficoll-Hypaque gradients activated with anti-CD3 and
were maintained in complete RPMI medium supplemented with 100 U/mL
recombinant interleukin-2 (rIL-2; EuroCetus, Milan, Italy). Forty-eight
hours after activation, 5 × 105 cells were transduced
with 1 mL lentiviral vector-containing supernatants for 6 hours at
37°C; subsequently, peripheral blood lymphocytes were transferred to
24-well plates (Becton Dickinson, Franklin Lakes, NJ) and were cultured
for an additional 72 hours before EGFP detection. In a set of
experiments, EGFP+ T lymphocytes were sorted by
fluorescence-activated cell sorter (FACS) and cultured in 96-well
plates in the presence of allogenic irradiated feeder cells
(105 cells/well) and IL-2 (100 U/mL) for 6 weeks.
Cytofluorometric analysis EGFP expression in vector-transduced cells was assessed on an EPICS-Elite cytofluorometer (Coulter, Hialeah, FL) by FACS analysis. At different times after infection, cells were pelleted, washed, fixed, and labeled, where appropriate, with a phycoerythrin-labeled anti-HLA class I monoclonal antibody (mAb) (DAKO, Glostrup, Denmark) or with a PC5-labeled anti-human CD3 mAb (Coulter). Two-color immunofluorescence was carried out as reported25 and was analyzed using the PRISM parameter of the Elite cytofluorometer; the negative control setting for each monoclonal antibody was determined by using labeled immunoglobulin of the corresponding isotype. Mock-transduced cell lines served as the negative control for EGFP-expression analysis. Mean fluorescence intensity (MFI) was calculated by the following formula: MFI = log10(mean × 10) × (1024/4). Coefficient of variation (CV) was calculated by the following formula: CV = [SD/mean] × 100; this parameter describes the homogeneity of the bell-shaped curve of fluorescence expression.In vivo expression studies Female severe combined immunodeficiency (SCID) mice (6 to 8 weeks old) were purchased from Charles River (Wilmington, MA). Logarithmically growing human Molt-3 cells transduced with the LESN or the LESN-LCR retroviral vectors were harvested and resuspended in
fresh medium at a density of 2 × 108 cells/mL. One
hundred microliters of this cell suspension was then injected into the
flanks of the mice. Tumor growth was monitored by periodic observation.
Mice were killed 1 month later, when the tumors were visible; tumor
cells were recovered by microdissection and were analyzed for HLA and
EGFP expression. At least 5 animals were used in each group, and the
experiment was repeated twice.
Generation of CD2 LCR-carrying retroviral vectors with unmodified LTRs We first amplified the 2.1-kb CD2 LCR sequence from the VA plasmid11 and cloned it in both orientations downstream of the EGFP cDNA in the LESN retroviral vector (Figure 1). Vectors generated were termed LESN-LCR and LESN-LCRas, and they carried the LCR in the sense or antisense orientation, respectively, relative to the EGFP gene. Retroviral vector-containing supernatants were generated and initially were used to transduce NIH-3T3 cells for determination of the titer of the vector stocks (Table 1). Transduction of Molt-3 cells with these vectors, followed by G418 selection, yielded a cell population expressing the EGFP marker at high levels. As shown in Figure 2, the pattern of EGFP expression did not differ between cells transduced with the vector carrying the LCR in either orientation compared with the parental LESN retroviral vector; a broad peak of reporter gene expression was detected in either case, as quantitatively indicated by the CV parameter at FACS analysis, which was comparable in the different fluorograms (Figure 2). These findings showed that CD2 LCR did not modulate EGFP expression when this sequence was placed in the context of a retroviral vector carrying full-length LTRs.
Generation of CD2 LCR-carrying retroviral vectors with modified LTRs To investigate LCR function in the absence of the viral enhancer, we generated a modified LESN retroviral vector carrying a deletion in the U3 region of the LTR and termed it LESN (Figure 1B). Preliminary
experiments indicated that its EGFP expression in transduced NIH-3T3
cells was strongly attenuated compared with LESN, in terms of MFI (data
not shown); this was expected because of the absence of the viral
enhancer. We next inserted the 2.1-kb LCR sequence in the LESN
vector in the antisense orientation (Figure 1B) and used this vector to
transduce lymphoid and nonlymphoid target cells. Furthermore, a 2.0-kb
sequence from the human CD2 gene, presumably devoid of LCR
activity, was also amplified by PCR from the VA plasmid (Figure 1A),
cloned in the LESN vector in the antisense orientation, and used as
a control for LCR function in some experiments. The vector carrying
this control sequence was termed LESN-NLCR (Figure 1B). Finally, to
study whether the full-length LCR was required to exert its
activity on gene expression, we constructed a retroviral vector
carrying a shorter fragment of the CD2 LCR containing a
T-lymphoid-specific enhancer, which was predicted to lack LCR
activity, and termed it LESN-LCR1 (Figure 1B).
We first measured the titer of the CD2 LCR-carrying vectors by transduction of NIH-3T3 cells, followed by G418 selection. Deletion of the viral enhancer or the inclusion of a truncated LCR sequence did not reduce viral titer compared with the parental LESN vector; however, all vectors carrying the full-length LCR or the control sequence generated viral titers that were on average 500-fold reduced compared with LESN (Table 1). Patterns of EGFP expression in lymphoid and nonlymphoid cells Molt-3 cells transduced by the different recombinant vectors and selected in G418-containing medium were simultaneously analyzed for EGFP expression 4 weeks after gene transfer. The percentage of EGFP+ cells in the different pools ranged from 70% to 100%, with some variability among the experiments and with the recombinant vector used (Table 2); prolonged in vitro culture for an additional 4 weeks did not further increase the fraction of EGFP+ cells, indicating that selection was complete at the time of analysis. We analyzed cells transduced by the LCR-containing vector and observed a unimodal pattern of expression, with MFI at an intermediate level (MFI = 551) between that generated by the LESN vector devoid of LCR (MFI = 504) and
the positive control LESN vector (MFI = 618) (Figure
3A). The vector carrying the control sequence failed to improve EGFP expression compared with the parental LESN vector; in fact, both percentage of EGFP+ cells and
MFI were reduced compared with cells transduced by the parental vector
lacking the viral enhancer (LESN), suggesting a negative
influence of the NLCR sequence on the expression of the reporter gene
(Figure 3A). Changes in the pattern of EGFP expression by LCR-carrying
vectors can be quantified by the CV parameter at FACS analysis; this
parameter describes the profile of marker gene expression by measuring
the width of the bell-shaped curve and, thus, fluorescence homogeneity.
The CV value was dramatically reduced in T-lymphoid cells transduced by
the LCR-carrying vector (CV = 30.4) compared with LESN-transduced
cells (CV = 107.8) (Figure 3A). Similar findings were obtained in 6 independent experiments, which are summarized in Table 2, indicating
that the CD2 LCR consistently modulated the EGFP expression pattern in
Molt-3 cells. These results were also confirmed by UV microscope
observation of the transduced cells; as shown in Figure 3B, the
LCR-carrying vector generated a homogeneous fluorescence pattern in
transduced Molt-3 cells compared with the highly heterogeneous
fluorescence profile generated by the parental LESN vector.
To investigate whether these effects on gene expression were specific
to Molt-3 cells, we transduced another T-cell line (Jurkat) and NIH-3T3
cells with the different retroviral vectors. As shown in Figure
4, infection of Jurkat cells with the
LCR-containing vector was associated with a profile of EGFP expression
fully comparable to that obtained in Molt-3 cells, and a unimodal
pattern of EGFP expression was observed (Figure 4). On the contrary,
the CD2 LCR was inactive in non-T cells because the LCR construct generated a similar pattern of EGFP expression in NIH-3T3 cells as the parental vector LESN
Finally, to determine whether the unimodal pattern of EGFP expression
observed required the full-length LCR element, we transduced Molt-3
cells with a retroviral vector carrying a truncated fragment of the LCR
sequence, which was expected to lack LCR activity and to retain the
enhancer function (Figure 1B). This vector yielded increased EGFP
expression compared with the LESN
Southern blot analysis of transduced cells To investigate whether the different patterns of EGFP expression observed in transduced cells might depend on nonspecific provirus rearrangements, genomic DNA samples from pools of transduced cells after G418 selection were analyzed by Southern blotting. We found that the CD2 LCR-carrying vector was not rearranged in Molt-3 transduced cells; digestion with KpnI restriction enzyme yielded a 5.5-kb band (Figure 6A, lane 4) that exactly matched the expected length of an unrearranged provirus. Genomic DNA from LESN-, LESN-, and LESN-LCR1-transduced cells
digested with KpnI and hybridized to the same probe yielded
bands of 3.6 kb, 3.4 kb, and 4.4 kb, respectively, corresponding to the
calculated sizes of the vectors (Figure 6, lanes 1-2). Furthermore,
quantitative Southern blot analysis demonstrated only limited
variations in the relative number of provirus copies per genome in each
sample (Figure 6).
Lack of unspecific rearrangements was also confirmed by PCR amplification of the same DNA samples with EGFP-specific primers and primers binding to different sites of the LTR of the vectors (data not shown). Overall, these experiments indicated that retroviral vectors carrying CD2 LCR sequences are genetically stable and do not undergo unwanted rearrangements in transduced cells. Evaluation of LCR activity by analysis of EGFP expression in single clones To confirm the changes in EGFP expression profile observed in bulk cultures of T lymphoid cells at the level of single cells, Molt-3 cells transduced by either the LESN or the LESN-LCR retroviral vectors
were cloned by limiting dilution; reporter gene expression was analyzed
by FACS at different time points and in the absence of G418 selective
pressure. As shown in Figure 7A,
LESN-transduced clones showed heterogeneous levels of EGFP expression
that ranged from very high (as seen in clones 3 and 17) to intermediate
(as in clones 8 and 12) and low or very low levels (as seen in clones 1 and 4). On the other hand, 6 of 6 analyzed clones of
LESN-LCR-transduced Molt-3 cells expressed EGFP at intermediate
levels, with a limited degree of interclonal variation of gene
expression (Figure 7A). When the transduced cell lines and the clones
derived from them were cultured for 120 days in the absence of G418 and
were checked for EGFP expression at regular intervals by FACS (Figure
7B), the transgene expression progressively declined over time in
LESN-transduced bulk cultures (Figure 7B). In addition, LESN-transduced
clones showed down-regulation of EGFP expression with time; in some
clones, reporter gene expression became heterogeneous after 30 to 60 days and was reduced to almost background levels by day 120 (Figure 7B). On the contrary, LESN-LCR-transduced Molt-3 cells and most clones maintained EGFP expression in vitro over the entire course of
the experiment (120 days) (Figure 7B), thus showing that CD2 LCR
confers an advantage in terms of duration of gene expression in vitro,
in the absence of selective pressure. Southern blot analysis of these
clones showed that they carried only one randomly integrated copy of
the vector genome (Figure 7C).
Effects of the LCR on gene expression in vivo To study expression patterns in vivo, Molt-3 cells were transduced in vitro with either LESN or LESN-LCR retroviral vectors, selected in G418-containing medium for 4 weeks, and implanted subcutaneously into SCID mice. Analysis of EGFP expression in the bulk
culture at the time of implantation revealed similar percentages of
EGFP+ cells (88.3% vs 91.6%) in LESN- or
LESN-LCR-transduced cells (Figure 8);
not surprisingly, however, in view of above data (Figures 3, 4),
LESN-transduced cells expressed EGFP at higher levels than LESN-LCR-transduced cells, as indicated by the different MFI values
generated by the 2 cell populations (MFI = 604 vs MFI = 508), but
with a less homogeneous profile, as indicated by the CV value (112.0 vs
80.5). After in vivo growth of Molt-3 cells for 30 days, the animals
were killed, and transduced cells were recovered and analyzed by FACS
for human HLA class 1 expression and EGFP expression. The HLA
antigen, expressed by this lymphoid cell line, was used as a marker of
human origin to restrict analysis of EGFP expression to Molt-3 cells
and to verify that expression of a cellular gene did not undergo
changes after in vivo growth of the cells. EGFP expression was strongly
reduced in LESN-transduced cells compared with preimplantation levels;
indeed, only 11.2% of the HLA class I+ cells expressed
detectable levels of the reporter gene, and the MFI was also greatly
reduced compared with in vitro levels (MFI = 419 vs MFI = 604)
(Figure 8). Conversely, in the case of LESN-LCR-transduced cells,
72.4% of the Molt-3 cells expressed EGFP at intensity levels comparable to preimplantation figures (MFI = 539 vs MFI = 508) (Figure 8). A more heterogeneous pattern of EGFP expression, as evaluated by the CV parameter, was observed in the samples analyzed after in vivo passage compared with figures generated by Molt-3 cells
transduced by the same vector and exclusively cultured in vitro; this
may suggest that LCR in vivo might only partially shield the transgene
from silencing. Interestingly, the down-modulation of gene expression
in vivo, as observed with LESN-transduced cells, was restricted to the
transgene, because no changes were detected in the level of expression
of the human HLA molecule on recovered Molt-3 cells compared with
preimplantation levels (Figure 8). Thus, we concluded that the CD2 LCR
could at least partially maintain its functions, also in vivo, by
preventing or slowing transcriptional silencing of the
transgene.
Modulation of EGFP expression by the CD2 LCR in primary T lymphocytes To evaluate the activity of the LCR in primary T cells, we attempted to transduce them with the different retroviral vectors used in this study. However, because of the poor titer of the LCR-carrying vector, we could not recover EGFP-expressing T cells using the Mo-MLV-based retroviral vectors. Therefore, we switched to lentiviral vectors that have been reported to transduce various types of primary cells, including T lymphocytes, with high efficiency and that might accept LCR elements with reduced loss of titers compared with retroviral vectors.26 Various recombinant EGFP-expressing lentiviral vectors carrying the CD2 LCR upstream of an internal MLV promoter were, therefore, generated (Figure 9A) and used to transduce anti-CD3-activated T cells. Preliminary gene transfer experiments on NIH-3T3 cells disclosed that all recombinant vectors generated infectious particles and that high-titer stocks of the LCR-containing vectors could be obtained (Figure 9A). Gene transfer in primary T cells was followed by cytofluorometric analysis of the transduced cells for CD3 and EGFP expression (Figure 9B). All vectors transduced the EGFP gene in T cells, and EGFP expression was detected in 0.1% to 16.7% of cells, depending on the vector used and on the blood donor; results of 5 independent experiments are listed in Table 3. The CD2 LCR did not significantly modify the pattern of EGFP expression when placed upstream of the intact MLV LTR within the lentiviral vector, as observed with retroviral vector-transduced lymphoid cells (Figure 9B, panels LTR and LTR-LCR). Furthermore, the lentiviral vector carrying a deletion in the viral enhancer showed greatly reduced EGFP expression in the target cells compared with the parental vector with intact promoter-enhancer sequences (Figure 9B, panels and LTR). Inclusion of the CD2 LCR 5'
upstream of the enhancer-deleted MLV LTR was associated with an
increase in EGFP expression, measured by the MFI value, mainly
resulting from a reduction in the EGFP+ fraction expressing
the marker at arbitrarily defined low levels (Figure 9B, panels and
-LCR). This phenomenon was observed in all experiments and was
translated into more homogeneous EGFP expression in the transduced T
lymphocytes.
To determine the effects of the CD2 LCR on long-term expression of the reporter gene in primary cells, we FACS sorted the EGFP+ T cells transduced by either the LCR-containing vector or the LTR vector and analyzed EGFP expression on the sorted population after 6-week in vitro culture. As shown in Figure 9C, this experiment indicated that both vectors underwent a similar degree of down-modulation of EGFP expression in long-term culture, independent of the presence of the CD2 LCR. Thus, we concluded that the CD2 LCR up-modulated gene expression in primary T cells but could not block the decline in EGFP expression in long-term in vitro cultures.
One of the major drawbacks of current gene transfer procedures is
that gene expression is often observed only transiently because of
transcriptional silencing of the transgene, even though stable gene
delivery can be achieved, at least with some viral systems, including
retroviral and lentiviral vectors.27 This limitation has
long been recognized, and possible solutions to the problem have been
suggested, including replacement of viral with cellular enhancers,
inclusion in the vector of LTRs from viruses that show a decreased
propensity to be silenced in stem cells, such as the murine stem cell
virus,28 and insertion of LCRs, matrix attachment sites,
and insulators.29-31 The discovery of We selected the human CD2 LCR, a sequence that has been studied in detail8-10 and that is potentially useful for improving gene expression in T-lymphoid cells. Kaptein et al34 also focused on the CD2 LCR and found that it was incapable of generating position-independent expression of the adenosine deaminase transgene delivered by retroviral vectors, but retroviral vectors with unmodified LTRs were used in this study. We observed that a deletion of the viral enhancer might be required to detect LCR activity in this context, and this finding might explain the discrepancies between the 2 studies. Our finding is also strengthened by previous work in transgenic mice indicating that LCR activity might be impaired by retroviral LTRs because of silencer elements.35,36 The main finding of our study is that the CD2 LCR sequence is able to
modulate gene expression specifically in T cells, and this translated
into a homogeneous, unimodal pattern of EGFP expression in cells
transduced by retroviral vectors carrying the LCR compared with
controls (Figures 3, 4). A T-cell-specific increase in EGFP expression, in terms of MFI, without significant changes in the pattern
of expression was also observed in T cells transduced by a retroviral
vector carrying a shortened sequence that maintained the CD2 enhancer
but was devoid of LCR activity (Figure 5). However, different figures
generated by vectors carrying full-length or shortened LCR sequences
might not be directly comparable because of the different design of the
2 vectors. Both the LCR and the enhancer effects were tissue
specific We observed that Molt-3 cells transduced by the LCR-containing vector had a lower MFI than cells transduced by the LESN retroviral vector. We attributed this to the fact that EGFP is driven by a cellular enhancer in the former and by a viral one in the latter. This might restrict the exploitation of this vector for gene therapy to pathologic conditions in which even intermediate levels of transgene expression suffice for a therapeutic outcome. In this regard, it is still unknown whether homogeneous expression of the therapeutic gene in transduced cells, albeit at intermediate levels, would be preferable to heterogeneous expression at higher levels. In vivo experiments, designed to test whether modulation in the pattern of EGFP expression observed in vitro would hold in vivo, disclosed that lymphoid cells transduced by a standard retroviral vector underwent marked reduction in EGFP expression in vivo, as predicted on the basis of previous studies with other cell types, including transduced skin fibroblasts.37 Interestingly, this reduction was specific to the transgene because the endogenous human HLA class I gene was not down-modulated in the same cells. This emphasizes the differences in gene expression between virally transduced and physiologically expressed cellular genes in our system. Strikingly, the CD2 LCR could prevent transgene silencing in most of the implanted lymphoid cells; the percentage of EGFP+ cells and the MFI were similar to those observed in cultured cells. However, EGFP was expressed with a broad range of intensity by ex vivo recovered cells, as opposed to the unimodal pattern of expression observed in cultured cells, thus suggesting that LCR-mediated shielding from silencing in vivo might be incomplete. A limitation to the exploitation of the retroviral vectors containing
LCR sequences is clearly their low titer. The reduction in titer, at
least in part, could depend on the increased vector size after the
insertion of long sequences (2 kb) from the CD2 locus; indeed, the
titer of the control vector, which does not carry any LCR and is
approximately the same size as LESN Inclusion of the CD2 LCR sequence in lentiviral vectors was followed by a less dramatic reduction in vector titer, and it was possible to use lentiviral vectors to transfer the CD2 LCR in primary T cells and to detect its effects on the EGFP expression pattern in the absence of G418 selection. The reason for the difference in performance of retroviral and lentiviral vectors carrying LCR sequences is unknown, but it has been observed by others.26 In our study, titration of the vectors by a real-time PCR assay targeted at the EGFP gene indicated similar amounts of proviral DNA in NIH-3T3 cells transduced by the LCR-containing retroviral and the lentiviral vector (data not shown). This suggests that the 2 vectors might be generated at similar levels by the packaging cells but that they differ in either ability to integrate the proviral DNA or to express the transgene. Inclusion of the CD2 LCR in lentiviral vectors led to a homogeneous pattern of EGFP expression in the primary T cells, but it did not counteract the down-modulation of reporter gene expression in long-term cultures, as we observed with the clones derived from leukemia cells. This could in principle represent a problem for the exploitation of these vectors in a clinical setting. However, the in vitro assay used might have some limitations in relevance to the in vivo behavior of the genetically modified cells. Thus, the issue of long-term expression in primary cells will likely be definitively addressed only by future studies in a murine transplantation model.
We thank A. Bank for providing gag-polgpt, D. Kioussis for the VA hCD2 plasmid, and K. Uzbela and R. Wagner for
ViG
Submitted October 24, 2000; accepted July 15, 2001.
Supported in part by Telethon (grant A-126), ISS-AIDS Project, MURST 60% and 40%, Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la Ricerca sul Cancro, Fondazione Città della Speranza, and National Research Council (PF Biotechnology). S.M. is the recipient of a fellowship from Fondazione Italiana per la Ricerca sul Cancro. V.T. is a recipient of an AIRC fellowship.
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: Stefano Indraccolo, IST-Viral and Molecular Oncology Section and Department of Oncology and Surgical Sciences, University of Padua, Via Gattamelata, 64-35128 Padua, Italy; e-mail: stefano.indraccolo{at}unipd.it.
1. Bestor TH. Gene silencing as a threat to the success of gene therapy. J Clin Invest. 2000;105:409-411[Medline] [Order article via Infotrieve]. 2. Dillon N, Grosveld F. Transcriptional regulation of multigene loci: multilevel control. Trends Genet. 1993;9:134-137[CrossRef][Medline] [Order article via Infotrieve]. 3. Higgs DR. Do LCRs open chromatin domains? Cell. 1998;95:299-302[CrossRef][Medline] [Order article via Infotrieve]. 4. Li Q, Harju S, Peterson KR. Locus control regions: coming of age at a decade plus. Trends Genet. 1999;15:403-408[CrossRef][Medline] [Order article via Infotrieve].
5.
Chang JC, Liu D, Kan YW.
A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression.
Proc Natl Acad Sci U S A.
1992;89:3107-3110
6.
Novak U, Harris EA, Forrester W, Groudine M, Gelinas R.
High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.
Proc Natl Acad Sci U S A.
1990;87:3386-3390
7.
Plavec I, Papayannopoulou T, Maury C, Meyer F.
A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells.
Blood.
1993;81:1384-1392 8. Festenstein R, Tolaini M, Corbella P, et al. Locus control region function and heterochromatin-induced position effect variegation. Science. 1996;271:1123-1125[Abstract]. 9. Lang G, Wotton D, Owen MJ, et al. The structure of the human CD2 gene and its expression in transgenic mice. EMBO J. 1988;7:1675-1682[Medline] [Order article via Infotrieve]. 10. Greaves DR, Wilson FD, Lang G, Kioussis D. Human CD2 3'-flanking sequences confer high-level, T cell-specific, position-independent gene expression in transgenic mice. Cell. 1989;56:979-986[CrossRef][Medline] [Order article via Infotrieve].
11.
Zhumabekov T, Corbella P, Tolaini M, Kioussis D.
Improved version of a human CD2 minigene based vector for T cell 12. Lake RA, Wotton D, Owen MJ. A 3' transcriptional enhancer regulates tissue-specific expression of the human CD2 gene. EMBO J. 1990;9:3129-3136[Medline] [Order article via Infotrieve]. 13. Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989;7:980-990[Medline] [Order article via Infotrieve].
14.
Markowitz D, Goff S, Bank A.
A safe packaging line for gene transfer: separating viral genes on two different plasmids.
J Virol.
1988;62:1120-1124
15.
Yee JK, Miyanohara A, LaPorte P, Bouic K, Burns JC, Friedmann T.
A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.
Proc Natl Acad Sci U S A.
1994;91:9564-9568
16.
Pear WS, Nolan GP, Scott mL, Baltimore D.
Production of high-titer helper-free retroviruses by transient transfection.
Proc Natl Acad Sci U S A.
1993;90:8392-8396 17. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual (2nd ed). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989. 18. Indraccolo S, Minuzzo S, Feroli F, et al. Pseudotyping of Moloney leukemia virus-based retroviral vectors with simian immunodeficiency virus envelope leads to targeted infection of human CD4+ lymphoid cells. Gene Ther. 1998;5:209-217[CrossRef][Medline] [Order article via Infotrieve]. 19. Ausubel FM. Current Protocols in Molecular Biology. New York, NY: Greene Pub Assoc and Wiley-Interscience; 1988.
20.
Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK.
Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.
Proc Natl Acad Sci U S A.
1993;90:8033-8037 21. Indraccolo S, Simon M, Hehlmann R, Erfle V, Chieco-Bianchi L, Leib-Moesch C. Genetic instability of a dinucleotide repeat-rich region in three hematologic malignancies. Leukemia. 1995;9:1517-1522[Medline] [Order article via Infotrieve].
22.
Wegner SA, Ehrenberg PK, Chang G, Dayhoff DE, Sleeker AL, Michael NL.
Genomic organization and functional characterization of the chemokine receptor CXCR4, a major entry co-receptor for human immunodeficiency virus type 1.
J Biol Chem.
1998;273:4754-4760 23. Schnell T, Foley P, Wirth M, Munch J, Uberla K. Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum Gene Ther. 2000;11:439-447[CrossRef][Medline] [Order article via Infotrieve]. 24. Wagner R, Graf M, Bieler K, et al. Rev-independent expression of synthetic gag-pol genes of human immunodeficiency virus type 1 and simian immunodeficiency virus: implications for the safety of lentiviral vectors. Hum Gene Ther. 2000;11:2403-2413[CrossRef][Medline] [Order article via Infotrieve].
25.
Indraccolo S, Mion M, Zamarchi R, et al.
B cell activation and human immunodeficiency virus infection, V: phenotypic and functional alterations in CD5+ and CD5 26. May C, Rivella S, Callegari J, et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature. 2000;406:82-86[CrossRef][Medline] [Order article via Infotrieve].
27.
Persons DA, Nienhuis AW.
Gene therapy for the hemoglobin disorders: past, present, and future.
Proc Natl Acad Sci U S A.
2000;97:5022-5024 28. Pawliuk R, Eaves CJ, Humphries RK. Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen. Hum Gene Ther. 1997;8:1595-1604[Medline] [Order article via Infotrieve].
29.
Bird AP, Wolffe AP.
Methylation-induced repression
30.
Emery DW, Yannaki E, Tubb J, Stamatoyannopoulos G.
A chromatin insulator protects retrovirus vectors from chromosomal position effects.
Proc Natl Acad Sci U S A.
2000;97:9150-9155
31.
Rivella S, Callegari JA, May C, Tan CW, Sadelain M.
The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites.
J Virol.
2000;74:4679-4687
32.
Tuan D, Solomon W, Li Q, London IM.
The "beta-like-globin" gene domain in human erythroid cells.
Proc Natl Acad Sci U S A.
1985;82:6384-6388 33. Rivella S, Sadelain M. Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing. Semin Hematol. 1998;35:112-125[Medline] [Order article via Infotrieve]. 34. Kaptein LC, Breuer M, Valerio D, van Beusechem VW. Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo. Gene Ther. 1998;5:320-330[CrossRef][Medline] [Order article via Infotrieve].
35.
Osborne CS, Pasceri P, Singal R, Sukonnik T, Ginder GD, Ellis J.
Amelioration of retroviral vector silencing in locus control region beta-globin-transgenic mice and transduced F9 embryonic cells.
J Virol.
1999;73:5490-5496
36.
McCune SL, Townes TM.
Retroviral vector sequences inhibit human beta-globin gene expression in transgenic mice.
Nucleic Acids Res.
1994;22:4477-4481
37.
Palmer TD, Rosman GJ, Osborne WR, Miller AD.
Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.
Proc Natl Acad Sci U S A.
1991;88:1330-1334
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  B. Hendrickson, D. Senadheera, S. Mishra, K. C. T. Bui, X. Wang, B. Chan, D. Petersen, K. Pepper, and C. Lutzko Development of Lentiviral Vectors with Regulated Respiratory Epithelial Expression In Vivo Am. J. Respir. Cell Mol. Biol., October 1, 2007; 37(4): 414 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Zhang, S. I. Thornhill, S. J. Howe, M. Ulaganathan, A. Schambach, J. Sinclair, C. Kinnon, H. B. Gaspar, M. Antoniou, and A. J. Thrasher Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells Blood, September 1, 2007; 110(5): 1448 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lavenu-Bombled, B. Izac, F. Legrand, M. Cambot, A. Vigier, J.-M. Masse, and A. Dubart-Kupperschmitt Glycoprotein Ib{alpha} Promoter Drives Megakaryocytic Lineage-Restricted Expression After Hematopoietic Stem Cell Transduction Using a Self-Inactivating Lentiviral Vector Stem Cells, June 1, 2007; 25(6): 1571 - 1577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lutzko, D. Senadheera, D. Skelton, D. Petersen, and D. B. Kohn Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes J. Virol., July 1, 2003; 77(13): 7341 - 7351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ramezani, T. S. Hawley, and R. G. Hawley Performance- and safety-enhanced lentiviral vectors containing the human interferon-{beta} scaffold attachment region and the chicken {beta}-globin insulator Blood, June 15, 2003; 101(12): 4717 - 4724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Gough and E. W. Raines Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector Blood, January 15, 2003; 101(2): 485 - 491. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
such as silencers, insulators,
and locus control regions (LCRs)
-globin LCR, which could be useful for increasing erythroid-specific
synthesis of this protein.
+) is
present in each vector genomic-RNA encoding construct. LCR, 2.1-kb CD2
locus control region; NLCR, 2.0-kb control sequence from the
CD2 gene; LCR1, 1-kb CD2 LCR fragment; neoR,
neomycin phosphotransferase gene. (gray box) Deletion of the viral
enhancer in the LTR; (arrow) orientation of the LCR element in each
vector.
packaging
sequences.
B cell subsets.
J Clin Immunol.
1993;13:381-388