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GENE THERAPY
From the Children's Hospital Los Angeles, University
of Southern California School of Medicine, Los Angeles; University of
Bordeaux, Bordeaux, France; Developmental Biology, Hospital for Sick
Children, Toronto, Ontario, Canada; University of Adelaide, Adelaide,
Australia; TIGET, Instituto Scientifico H.S. Raffaele, Milan, Italy;
Department of Biomedical Sciences, University of Modena School of
Medicine, Modena, Italy; and Academic Sinica, Nankang, Taipei, Taiwan,
Republic of China.
Use of oncoretroviral vectors in gene therapy for
hemoglobinopathies has been impeded by low titer vectors, genetic
instability, and poor expression. Fifteen self- inactivating (SIN)
lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus
postregulatory element (WPRE) were generated using the enhanced green
fluorescent protein as a reporter gene. Vectors with high
erythroid-specific expression in cell lines were tested in primary
human CD34+ cells and in vivo in the murine bone marrow
(BM) transplantation model. Vectors containing the ankyrin-1 promoter
showed high-level expression and stable proviral transmission. Two
vectors containing the ankyrin-1 promoter and 2 erythroid enhancers
(HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the
HS2/ Gene therapy for red blood cell (RBC) disorders,
particularly for hemoglobinopathies, using retroviral vectors has been
fraught with problems. Moloney murine leukemia virus (MLV) vectors are the most extensively studied oncoretroviral vectors. However, they do
not transduce the nondividing hematopoietic stem cells. Exogenous genes
in MLV vectors, driven by the viral long terminal repeat (LTR)
promoter/enhancer, are expressed in a lineage-nonspecific manner. In
addition, the MLV LTR interferes with the erythroid lineage-specific
regulatory elements inserted in the vectors, resulting in unstable
proviral transmission or poor transgene expression or
both.1-3 Transcriptional interference from the LTR4-6 can be overcome by self-inactivating (SIN) vectors.
In SIN vectors, the LTR promoter/enhancers are deleted on integration of the provirus.7,8 However, SIN-MLV vectors have shown
limited applicability, because the deletion of the TATA box in them
results in inefficient 3' end processing of the viral RNA genome,
resulting in low titer vectors.
Retroviral vectors are RNA-based vectors and, therefore, are restricted
to incorporating complementary DNA (cDNA) forms of processed messenger
RNA (mRNA). This results in low expression of intron-dependent genes,
such as globin.9,10 Introns in the globin gene
enhance transcription and allow proper 3' end processing and export of
globin transcripts into the cytoplasm.11-14 Retention of
introns of the globin genes can be achieved by using vectors that
contain the globin gene in reverse orientation to the viral
transcriptional unit. However, this has not improved expression because
these vectors have additional problems with antisense effects of the
transcripts, low titers, and proviral instability.2
Another way is to increase the expression of the globin cDNA that can
be placed in sense orientation in the vector. Recently, the human and
woodchuck hepatitis virus posttranscriptional regulatory elements (HPRE
and WPRE) have been reported to replace the functions of the globin
intron and improve expression of the globin cDNA.15,16
The recently developed lentiviral vectors (derived from the human
immunodeficiency virus 1 [HIV-1])17 are attractive for globin gene therapy for several reasons. Lentiviral vectors have been
shown to infect nondividing cells, including the quiescent hematopoietic stem cells.18,19 Lentiviral LTR
transcription is tat dependent and lack of tat in the provirus results
in very low level transcription from the LTR. The low-level LTR
transcription is completely eliminated in SIN-lentiviral
vectors,20,21 making them ideal for lineage-specific gene
expression. Unlike SIN-MLV vectors, SIN-lentiviral vectors can be
packaged without loss of titers.20-22
We therefore hypothesized that modular use of erythroid-specific
enhancers and promoters and inclusion of the WPRE in SIN-lentiviral vectors could address issues of enhancer interference and intron independence of the globin gene and result in high-level
erythroid-expressing cassettes for gene therapy of RBC disorders. We
tested 4 erythroid enhancers with 4 erythroid promoters in different
combinations with and without the WPRE, using green fluorescence
protein (GFP) as the reporter gene. We observed a high-level, stable,
and erythroid-lineage specific gene expression from vectors containing
erythroid-specific elements. The highest levels of expression were seen
with the ankyrin-1 promoter, in combination with 2 enhancers in tandem (the GATA-1 and HS-40 or the I8 and HS-40 enhancer pairs) in primary human cells as well as in mice 11 weeks after bone marrow
transplantation (BMT), at levels higher than those observed with the
HS2 enhancer/ Vector construction
The cloning strategy for the
The BamHI/NotI fragment containing the
enhanced GFP cDNA (Clontech Laboratories, Palo Alto, CA) was inserted
into the BamHI/NotI sites of pS2 downstream of
the The Production and titration of vectors
DNA analysis Genomic DNA from transduced cells (murine erythroleukemia [MEL] and 293A) was analyzed by semiquantitative PCR for GFP to determine the proviral copy number as described previously.16 Genomic DNA Southern blot was performed on MEL cells to test integrity of the provirus by digestion with BamHI and XhoI for H2BGW-transduced cells and with EcoRI and XhoI for all other vector-transduced cells. Digested DNA was gel fractionated on 0.8% agarose gels, blotted, and hybridized with 32P-labeled GFP cDNA probe.RNA analyses To compare human -globin expression to endogenous mouse
 -globin RNA, RNAse protection assays (RPAs) were performed on total RNA extracted from transduced and differentiated MEL cells using the
Riboquant In Vitro Transcription Kit (Pharmingen, San Diego, CA)
according to the manufacturer's protocol. The linearized templates of
plasmids protecting a 280-bp human -globin mRNA fragment and a
113-bp mouse -globin coding sequence were used to transcribe 32P-UTP-labeled RNA in vitro (unprotected probe sizes were
309 bp and 142 bp, respectively). Cellular RNA was extracted using
RNA-stat and hybridized with an excess of 32P-UTP-labeled,
in vitro transcribed RNA probe and then subjected to RNAse digestion.
Protected fragments were resolved on a 6.5% polyacrylamide gel and
quantified by phosphoimager analyses. A very small amount of the
unprotected in vitro transcribed RNA (1/100 of the amount hybridized
with cellular RNA) was loaded on the gel.
Quantification of mRNA levels The level of the-globin mRNA was determined using a
phosphoimager (Biorad, Hercules, CA). The relative amounts of
emissions from the human -globin (hu-) RNA band and mouse
-globin (mu-) RNA bands were calculated as percentages by the
following formulas:
/total mouse  -globin from the HS3
enhancer/-promoter was used as a positive
control.29
Flow cytometry analyses Fluorescence-activated cell sorter (FACS) analysis for GFP was performed on a FACS-Calibur and FACS-Vantage flow cytometer (Becton Dickinson, San Jose, CA). A ratio of the mean fluorescence intensity (MFI) of transduced cells and nontransduced cells was used to normalize GFP expression by FACS. Comparative analyses of fluorescence intensity were performed on the same FACS machine with regular calibration standards and constant voltage for each cell line. For analysis of lineage-specific GFP expression in mice, cells were stained with phycoerythrin (PE)-conjugated antimouse antibodies to CD3, B220, Gr-1, and Ter-119 (Pharmingen). Antimouse CD45.2PE (Ly5.1, Pharmingen) antibody was used for evaluation of donor-host chimerism in bone marrow (BM), spleen, and thymus. Human cell phenotyping was performed using PE-conjugated antihuman CD13 (Pharmingen) and glycophorin A (GlyA; Pharmingen) antibodies.Cell lines and transduction The 293T and 293A cells (ATCC) and MEL cells were maintained in Dulbecco modified Eagle medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% horse serum (Omega Scientific, Tarzana, CA). MEL cells were induced to differentiate in DMEM, 10% fetal calf serum (FCS) containing 5 mM N, N-hexamethylene bisacetamide (HMBA, Sigma) and GFP expression determined by flow cytometry at day 4 of differentiation. The human K562 cells were maintained in RPMI (Gibco) supplemented with 10% FCS. All cell lines were transduced with serial dilutions of viral supernatant ranging from a multiplicity of infection (MOI) of 0.1 to 10, in the presence of 4 µg/mL polybrene (Sigma). On day 7 following transduction, cells were harvested and analyzed by FACS for GFP expression.Hematopoietic cells and transduction The CD34+ progenitor cells were isolated from normal human BM mononuclear cells using Mini-MACS columns (Miltenyi Biotech, Auburn, CA). Use of samples was approved by the Committee on Clinical Investigations at Children's Hospital Los Angeles. CD34+ cells (5 × 104) were suspended in 100:l of X-vivo 15 medium (Biowhittaker, Walkersville, MD) containing the following recombinant cytokines: recombinant human (rh) interleukin (IL)-3 (rhIL-3; 10 ng/mL), rhIL-6 (10 ng/mL), rhFlt-3 (10 ng/mL), rh thrombopoeitin (rhTPO; 10 ng/mL), and rh stem cell factor (rhSCF; 25 ng/mL) and in 96-well plates coated with CH-296 fragment of recombinant fibronectin (Retronectin, Takara Shuzo, Otsu, Japan). Lentiviral supernatants at an MOI of 10 were added twice daily, 12 hours apart, after overnight prestimulation in the above medium. On day 2, transduced CD34+ cells were washed in phosphate-buffered saline (PBS) and used for in vitro assays.Colony-forming unit assays The CD34+ cells (500, 1000, and 1500) were plated in duplicate in methyl cellulose containing rhSCF (50 ng/mL), rhIL-3 (10 ng/mL), rhIL-6 (20 mg/mL), rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.01 ng/mL), and rh erythropoietin (rhEpo; 3 U/mL) to support erythroid burst-forming units (BFU-Es) and the same combination of cytokines, but with 10 mg/mL rhGM-CSF without Epo, to support granulocyte-macrophage colony-forming units (CFU-GMs). BFU-Es and CFU-GMs were scored 14 days after plating, then harvested, washed with PBS and analyzed for GFP expression by FACS.In vitro differentiation into unilineage erythroid and myeloid cells The CD34+ cells were placed in erythroid culture conditions in basal BM medium (BBMM; IMDM based, supplemented with 25% FCS) with IL-3, GM-CSF, and Epo, as previously described30 or in myeloid differentiation conditions (BBMM with huGM-CSF [10 ng/mL] huSCF [25 ng/mL] huIL-3 [10 ng/mL],) and huIL-6 [10 ng/mL]). Erythroid and myeloid cultures were harvested after 2 weeks and analyzed for GFP expression and erythroid or myeloid surface markers by FACS.Transduction of murine BM and BMT Bone marrow was harvested from C57Bl/LY5.1 mice (B/6.SJL-CD45a-Pep3b, Jackson Laboratory, Bar Harbor, ME) (CD45.1+) by flushing femurs and tibiae and sorted for Sca-1+ cells using the MACS Sca-1 multisort kit (Miltenyi Biotech). The Sca-1+ cells obtained were lineage-depleted by staining with a lineage cocktail of fluorescein isothiocyanate (FITC)-labeled antibodies to murine CD3, CD4, CD8, CD11b, Gr-1, and Ter-119 and PE-labeled antibody to Sca-1. The Sca+Lin cells were sorted by FACS and were
transduced in 1 mL Stemspan (Stemcell Technologies, Vancouver,
BC, Canada) supplemented with 20% FCS containing 10 ng/mL of each of
the following cytokines: muTPO, huFlt-3, muIL-3, huIL-6, muSCF, 4 U/mL
hu-Epo, 10 mM dNTP, and 40 mg/mL low-density lipoprotein (LDL;
Sigma). Cells were transduced with viral supernatants from the HTKGW,
HI8KGW, or H2BGW lentiviral vectors 3 times at 12-hour intervals after
overnight prestimulation, using similar concentrations of virus at
5 × 106 transmitted units (TU)/mL (MOI 100). On
day 3, cells were washed twice with PBS and 15 000 hematopoietic
cells/mouse were injected into the tail vein of 8- to 12-week-old male
C57BL/6-ly5.2 (CD45.2+) mice (Jackson Laboratories), that
had been irradiated with 12 Gy in 2 split doses of 6 Gy, 24 hours
apart. Eleven weeks after BMT animals were killed and peripheral blood,
BM, thymus, and spleen were harvested. In blood, RBCs were analyzed
before RBC lysis for white blood cell (WBC) analyses. FACS analysis was
performed for donor cells (CD45.2), GFP expression in different
lineages (T cells, B cells, granulocytes, and RBCs), and DNA analysis
to determine proviral copy number.
Generation and titration of SIN-lentiviral vectors To develop a high-expressing, erythroid-specific SIN-lentiviral vector, a series of 15 SIN-lentiviral vectors containing different modular combinations of erythroid promoters and enhancers were constructed (Figure 1A-E). The 4 erythroid promoters from the erythroid-specific genes ankyrin-1,-spectrin,
the  -globin (in the context of HS-40), and the
-globin were tested. The cytomegalovirus (CMV)
promoter vector served as a lineage-nonspecific control. The HS-40
enhancer used in these studies contains a single base pair mutation at
the NFE2 site that results in derepression of the -globin
promoter in adult erythroid cells and confers position-independent expression.25 Other erythroid enhancers used were the
GATA-1 ARE, I8 from the eALAS gene, and HS2 from the
-globin locus control region (LCR). The -spectrin and
ankyrin-1 promoter series of vectors were tested with and without
the WPRE.
Viral titers were determined by transducing 293A cells with serial dilutions of concentrated virus and determining the number of transmitted copies per cell by semiquantitative PCR. Due to extreme erythroid specificity of the vector cassettes, determination of titers by quantifying the proportion of GFP-expressing 293A cells was not possible. Vector titers ranged from 2.2 × 107 to 1.1 × 108 TU/mL, with a mean titer of 4.5 × 107 TU/mL, after a 100-fold concentration by ultracentrifugation. Thus, high-titer SIN-lentiviral vectors, containing various internal erythroid promoter/enhancers, could be obtained. All vectors transmitted an intact provirus, except 2 vectors in the
Screening for vectors with strong erythroid activity in cell lines We first screened all SIN-lentiviral vectors in 6 different erythroid and nonerythroid cells lines (Figure 2A-C). Data from the nonerythroid human kidney 293A cells and the erythroid K562 cells (that express fetal-type globin) from 4 independent experiments is shown in panels A and B of Figure 2. K562 cells showed no remarkable difference in GFP expression after erythroid differentiation with hemin (40 µM) and sodium butyrate (100 µM) and, therefore, only data from undifferentiated cells are shown. Expression in MEL cells, which express adult-type globin on differentiation, was more clinically relevant and is shown before and after differentiation (Figure 2C). Because vectors expressing at high level in MEL cells were primarily of interest, semiquantitative PCR analysis was performed on transduced populations to normalize GFP expression level for proviral copy number. A representative experiment is shown in Figure 2C.
To favor single proviral integration/cell, all of these experiments were done at a relatively low MOI (MOI of 0.1 for the adherent 293A cells, and MOI of 1 for the nonadherent K562 and MEL cells) and the comparative FACS analyses were performed on cell pools with less than 30% transduction efficiency. The ratio between the MFI of GFP in transduced and nontransduced cells was used to quantify promoter/enhancer strength. Although all 3 cell lines transduced by the control CMV lentiviral
vector CMV-G expressed GFP (Figure 2A-C), expression from vectors
containing erythroid elements was observed exclusively in K562 (Figure
2B) and MEL cells (Figure 2C). In K562 and MEL cells, expression from
vectors containing the ankyrin-1 promoter was higher (1.5-fold and
6-fold, respectively) than that from the Overall, vectors containing the ankyrin-1 promoter expressed at highest levels in erythroid cell lines and the expression was inducible on MEL cell differentiation. The presence of 2 erythroid enhancers increased expression and inclusion of WPRE nearly doubled GFP expression. Expression of erythroid SIN-lentiviral vectors in primary human BM cells Although MEL and K562 cell lines were useful for an initial evaluation of transgene expression, comparison of transgene expression in primary human BM progenitors during their erythroid differentiation was more relevant. Therefore, the best 10 vectors from the initial screening were tested in primary cells. Human CD34+ cells from 4 different BM samples were transduced at an MOI of 10. This MOI favors single integration per cell in primary human CD34+ cells in suspension cultures. A portion of transduced CD34+ cells was plated for colony-forming assays and the remainder grown in liquid cultures for 2 weeks, in unilineage conditions favoring erythroid or myeloid differentiation. At 2 weeks, GFP-expressing colonies were scored and picked for FACS analyses. Cells in liquid culture were stained with PE-conjugated anti-GlyA or anti-CD13 antibodies and analyzed by FACS.Table 1 shows that both erythroid and
myeloid colonies transduced with CMV-G vector expressed GFP at a
similar frequency. In contrast, with all other vectors expression of
GFP was only observed in BFU-Es alone, demonstrating a particularly
high lineage specificity of these vectors. Gene transfer, based on GFP
expression, was similar in all vectors, ranging from 18% to 29%.
To compare the level of GFP expression from each vector, at least
10 randomly selected BFU-Es were pooled per experiment and were
analyzed by FACS (Figure 3A, n = 4).
The highest MFI (mean ± SD) was observed in BFU-Es transduced
with the HTKGW vector and was significantly higher than expression in
BFU-Es transduced with the H2BGW, CMV-G, or HKGW vectors
(P < .05). This was closely followed by expression from
the HI8KGW vector. Consistent with the cell line data, BFU-Es
transduced with the
The NFE2 mutation HS-40 has been shown to allow position-independent expression of transgenes in adult erythroid cells.25 Therefore, we studied gene expression at a clonal level, by analyzing 40 individual BFU-Es per vector by FACS in 4 individual experiments (Figure 3B). The level of GFP expression was variable in the presence of the HS-40 as well as the HS2 LCR element. This variability may be attributed to differences in maturation of individual BFU-Es or a relative lack of position independence. The mean MFI data on the individual BFU-Es closely paralleled that from pooled BFU-Es. Panel C in Figure 3 shows a FACS analysis of myeloid and erythroid
liquid cultures of the representative vectors. The results are
consistent with the colony data. The CMV-G vector expressed in the
myeloid as well as the erythroid progeny of CD34+ cells,
whereas the erythroid vectors expressed only in the GlyA+
cells. High levels of GFP expression were evident from the H2BGW, HTKGW, and CMV-G vectors and the MFI from In vivo activity of SIN-lentiviral vectors We next studied the in vivo expression of the 3 best vectors, H2BGW, THKGW, and I8HKGW, in the murine BMT model. BM Sca+Lin cells from Ly5.1 donor mice were
transduced with the HTKGW, I8HKGW, and H2BGW vectors and transplanted
into lethally irradiated Ly5.2 recipient mice. Eleven weeks
after BMT, recipient mice were killed and blood, BM, spleen, and thymus
were studied for GFP expression in the erythroid (TER119), myeloid
(Gr1), T lymphocyte (CD3), and B lymphocyte (B220) lineages. The
engraftment of the Sca+Lin donor
cells (Ly5.1) was high with a mean of 70% ± 9.7% (mean + SD)
in BM and 85.9% ± 9% in peripheral blood. The expression of all 3 vectors was restricted to the erythroid lineage in BM, thymus, spleen,
and peripheral blood in all mice (n = 8), consistent with results
seen in cell lines and primary human cells.
Figure 4 shows data from 3 mice that had
comparable BM engraftment (84%-92% donor cells) and similar proviral
copy numbers (2-2.5 copies/cell). We observed a very high frequency of
RBCs expressing GFP from H2BGW, THKGW, and I8HKGW vectors in peripheral blood, ranging from 63% to 87% (Figure 4A). After RBC lysis, GFP expression was analyzed in WBCs. Minimal GFP expression was seen in B,
T, and myeloid cells (0.5%-3.7%) from all 3 vectors. Similar lineage
specificity of GFP expression was observed in spleen, thymus, and BM
cells, despite the presence of proviral DNA (data not shown).
We next analyzed GFP expression in BM erythroid cells at
progressively increasing stages of erythroid differentiation, by gating
on large (early normoblasts), intermediate (intermediate normoblasts),
and small cells (RBCs), based on their forward cell scatter (FCS)
profile and Ter-119 expression (Figure 4B, panels I-III). There was
negligible expression of GFP in the TER119 The MFI was higher in the more immature erythroid cells and decreased 2- to 3-fold with maturation with all 3 vectors, probably due to smaller cell size and shorter half-life of GFP in the transcriptionally inactive, enucleated RBCs. Progressive hemoglobinization with maturation may also result in quenching of GFP fluorescence because hemoglobin absorption and GFP emission wavelengths are similar. However, at all stages of maturation, the mean intensity from the ankyrin-1 promoter vectors (THKGW and I8HKGW) containing either of 2 enhancer element pairs is higher than that from the H2BGW vector. Expression of human W vector
at low MOI (MOI 3-10) to obtain cell pools with approximately
one copy per cell. Human -globin expression was analyzed in several
unselected pools of cells. The proviral copy number of 3 cell pools,
determined by semiquantitative PCR analyses, were 1.26, 1.9, and 1.25 copies/cell. Figure 5 shows an RNAse
protection assay on the sham-transduced and
I8HW-transduced MEL cells on differentiation. Human
-globin expression/total murine -globin in these pools was 35.5%
(lane 2), 24% (lane 3), and 14% (the mean expression from duplicate samples in lanes 4 and 5), respectively. When normalized to single vector copy, this translates to 11% to 28% human -globin/total murine -globin or 43% to 113% human -globin/copy of murine
-globin or 22% to 56% human -globin/murine -globin
allele.
We tested SIN-lentiviral vectors in cell lines, in primary human
cells, and in the murine BMT model. In all 3 systems (1) expression
from vectors containing erythroid promoters and enhancers was highly
lineage specific; (2) expression from the ankyrin-1 promoter vectors
was higher than that from the High-level erythroid-specific expression in primary human and murine BM cells from SIN-lentiviral vectors could be obtained due to the absence of cis-acting influences from the HIV LTR promoter/enhancer. The lentiviral LTR is relatively inactive in the absence of tat allowing photoreceptor cell-specific expression, as has been previously reported in rat retina using the rhodopsin promoter.31 However, these investigators later reported that an internal promoter placed within the lentiviral vector resulted in transcriptional interference that was overcome with deletion of the 3' LTR enhancer in the SIN vector.21 The Replacement of the MLV LTR enhancer by the GATA-1 ARE enhancer has been
previously reported to confer erythroid-specific expression to MLV
vector cassette.23 GATA-1 ARE showed lower enhancement of
expression than HS-40. However, if inserted in tandem with the HS-40,
it enhanced expression levels to levels higher than the HS2/ The HS2 from the The The human ankyrin-1 gene promoter was also chosen for
similar reasons. Transgenic mice carrying the 272-bp core ankyrin-1 promoter fused with the The HPRE and WPRE have been reported to replace 2 of the known
functions of The high level of erythroid-specific GFP expression is also translated
to expression of therapeutic genes. We observed 43% to 113% human
In summary, previous studies in gene therapy for hemoglobinopathies
using oncoretroviral vectors using different erythroid regulatory
elements have had problems with vector instability, interference from
the viral LTR, low titer vectors of SIN-MLV vectors, and unpredictable
expression of globin. Use of the SIN lentiviral vectors that lack LTR
transcription allowed development of erythroid lineage-specific
vectors. It also allowed comparisons of strengths of different promoter
and enhancer combinations that result in stably transmitted high-level
expression. Whether these vectors will resist transgene silencing in
the long-term in vivo or require the use of insulator elements is
currently under study. Further, vectors incorporating
We would like to thank Drs Inder Verma and H. Miyoshi for providing
the SIN lentiviral vector backbone, Dr Philippe Leboulch for
providing the HS2/
Submitted August 25, 2000; accepted June 10, 2001.
Supported by the Association pour la Recherche sur le Cancer, Villejuif, France, the John Connell Gene Therapy Program, Children's Hospital Los Angeles, and the Sickle Scholar Award, University of Southern California Comprehensive Sickle Cell Center grant HL96-002B.
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: Punam Malik, Division of Hematology Oncology, Children's Hospital Los Angeles, MS 54, 4650 Sunset Blvd, Los Angeles, CA 90027; e-mail: pmalik{at}chla.usc.edu.
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Top
Abstract
Introduction
509 to +203) from the transcriptional start site, with ClaI and BamHI ends
P8.91 (10 µg), VSV-G pMD.G (5 µg), and a vector construct (15 µg), induced with sodium butyrate (Sigma, St Louis, MO)
and viral supernatant was collected and concentrated by
ultracentrifugation.
![[total mu &agr;-globin RNA (4 &agr;-globin genes)]×100.](/content/vol98/issue9/fulltext/2664/img002.gif)
RBC), intermediate (II