|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
GENE THERAPY
From the Institut National de la Santé
et de la Recherche Médicale (INSERM) U362, Institut Gustave
Roussy, Villejuif, France, and the Unité d'Oncologie Virale,
Institut Pasteur, Paris, France.
Gene transfer in human hematopoietic stem cells (HSCs) has great
potential for both gene therapy and the understanding of hematopoiesis.
As HSCs have extensive proliferative capacities, stable gene transfer
should include genomic integration of the transgene. Lentiviral vectors
are now preferred to oncoretroviral vectors especially because they
integrate in nondividing cells such as HSCs, thereby avoiding the use
of prolonged cytokine stimulation. Human immunodeficiency virus type-1
(HIV-1) has evolved a complex reverse transcription strategy including
a central strand displacement event controlled in cis by the central
polypurine tract (cPPT) and the central termination sequence (CTS).
This creates, at the center of HIV-1 linear DNA molecules, a
99-nucleotide-long plus-strand overlap, the DNA flap, which
acts as a cis-determinant of HIV-1 genome nuclear import. The
reinsertion of the DNA flap sequence in an HIV-derived lentiviral
vector promotes a striking increase of gene transduction efficiency in
human CD34+ hematopoietic cells, and the complementation of
the nuclear import defect present in the parental vector accounts for
this result. In a short ex vivo protocol, the flap-containing vector
allows efficient transduction of the whole hierarchy of human HSCs
including both slow-dividing or nondividing HSCs that have multiple
lymphoid and myeloid potentials and primitive cells with long-term
engraftment ability in nonobese diabetic/severe combined
immunodeficiency mice (NOD/SCID).
(Blood. 2000;96:4103-4110) Human hematopoietic stem cells (HSCs) have the
potential to regenerate the entire hematopoietic system, and as such,
they are the target cells both for gene therapy and the understanding of mechanisms regulating normal and pathological hematopoiesis. As HSCs
have extensive proliferative capacities, stable gene transfer should
include genomic integration of the transgene. Retroviral vectors based
on Moloney murine leukemia virus (MoMuLV) have been very popular
because they systematically integrate their genome into the host cell
chromatin. However, like the wild-type virus, MoMuLV-derived vectors do
not integrate in nondividing cells including human HSCs that are almost
all quiescent. Many prestimulation protocols using different cytokine
combinations have been developed to trigger cell cycling of HSCs, and
thus these protocols induce HSC susceptibility to transduction
by the oncoretrovirus-derived, mitosis-dependent gene-transfer vectors.
However, prolonged cytokine stimulation can induce differentiation
together with proliferation, thereby leading to the loss of fundamental
properties of HSCs during the transduction process. The use of
prolonged cytokine stimulation to trigger cell cycling can be overcome
by using lentivirus-derived vectors because lentiviruses have evolved a
mitosis-independent nuclear import strategy that accounts for their
unique capacity, among retroviruses, to replicate efficiently in
nondividing target cells.2,3
The search for the viral determinants responsible for the active
nuclear import of the human immunodeficiency virus type-1 (HIV-1)
complementary DNA (cDNA) genome is an active but controversial field of
investigation.4-12 HIV-1 has evolved a complex reverse transcription strategy including a central strand displacement event
controlled in cis by the central polypurine tract (cPPT) and the
central termination sequence (CTS).13-16 Reverse
transcription results in a linear DNA molecule bearing in its center a
stable 99-nucleotide-long plus-strand overlap, here referred to as the central DNA flap. We have previously established that the central DNA
flap of HIV-1 is involved in a late step of HIV-1 genome nuclear import, immediately prior to or during viral DNA translocation through
the nuclear pore.1 This result has important implications for the design of efficient lentiviral vectors because it is
essential to maintain the lentiviral nuclear import determinants within vector constructs. Classical retroviral vector constructs are replacement vectors in which the entire viral coding sequences between
the 2 long terminal repeats (LTRs) are replaced by the sequences of
interest.17 In the case of lentiviral vectors, this leads
to the deletion of the central cis-active cPPT and CTS sequences,
which suggests that such replacement vector constructs are not optimal.
We show here that introduction of the DNA flap sequence in an
HIV-derived lentiviral vector,18 the resulting vector being called TRIP vector, dramatically increased gene transduction efficiency in human CD34+ hematopoietic cells by strongly stimulating
nuclear import of the vector pre-integration complex. Most importantly,
we show that during a 24-hour ex vivo transduction protocol, an
efficient and equivalent transduction of the whole hierarchy of human
HSCs is indeed achieved. The transduced cells include slow and
nondividing HSCs, which have multiple lymphoid and myeloid potential,
and primitive cells, which are able to establish long-term human
hematopoiesis in nonobese diabetic/severe combined immunodeficiency
(NOD/SCID) mice.
Collection and fractionation of cells
Lentiviral vector supernatants
Transduction protocol
Lentiviral vector particles were added at a concentration corresponding
to 100 ng viral P24/mL (multiplicity of infection, Hematopoietic cell cultures Colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) were assayed as described.24,25 Lymphoid (B and natural killer [NK]) and myeloid (granulomonocytic and erythrocytic) potentials were assessed as described19 on MS5 stromal cells in the presence of rhSCF; rhFL; rhPEG-MGDF; and rhIL-2, rhIL-7, and rhIL-15. The cytokine cocktail also contained 10 ng/mL rhIL-3 and, in some experiments, 2 U/mL rhEPO (erythropoietin) (CILAG, Issy les Moulineaux, France). In all cases, cells were analyzed for EGFP expression by flow cytometry and phenotyped using the following mouse monoclonal antibodies (mAbs): CD19-PE (phycoerythrin) (Becton Dickinson), CD15, CD14, GPA-PE (PharMingen, Pont de Claix, France), CD11b-PE, CD56, and CD34-PE-Cy5 (Immunotech, Villepinte-Roissy CDG, France). Unspecific staining was detected using irrelevant mouse immunoglobulin G1 (IgG1) and IgM mAbs.PCR analysis Integration of the EGFP transgene was analyzed by PCR analysis on DNA extracted from CFC-derived colonies and from cells in lymphomyeloid cultures. DNA was prepared as previously described.26 Amplification of genomic DNA was performed on the entire extract with primers that amplify part of the vector-encoded EGFP sequence (5'-CCCTCGAGCTAGAGTCGCGGCCG-3' sense primer and 5'-CCGGATCCCCACCGGTCGCCACC-3' antisense primer). The amplification was performed for 35 cycles at an annealing temperature of 62°C, resulting in an 800-bp PCR product.Assessment of the number of cell divisions CD34+ cells were stained with PKH26 (Sigma-Aldrich) according to the manufacturer's instructions. Immediately after labeling, a CD34+ cell population corresponding to a narrow band of PKH26bright cells was isolated by cell sorting27 and transduced as described above. An aliquot of these cells was cultured in parallel in the presence of 0.1 µg/mL colcemid (Demecolcemid, Sigma-Aldrich) to serve as an internal control of nondivided cells. Fluorescence of PKH26-labeled transduced cells was analyzed by flow cytometry at the end of the 24-hour transduction protocol as well as after an additional 48 hours of culture in lymphomyeloid conditions. At that time cells were analyzed for both EGFP expression and PKH26 staining. In 2 experiments cells having undergone 0 or 1 division were sorted, and single cell clones were cultured in LTC and lymphomyeloid conditions. Sorting gates were defined in comparison with colcemid-treated cells.Transplantation of CD34+ cells into NOD/SCID mice Immediately after transduction, 7-10 × 104 cells were intravenously injected into sublethally irradiated NOD-LtSz-scid/scid (NOD/SCID) mice (3-3.5 Gy at 0.43 Gy/mn in an x-ray Phillips RT250 irradiator). Fifteen weeks later, BM cells were harvested from recipient mice, and the presence of human cells was assessed in individual mice by flow cytometry using the following mouse antihuman mAbs: CD38-PE, CD19-PE, CD34-PE-Cy5, and CD14-PE. EGFP expression was also analyzed in each differentiated population. Human CD34+CD38 cells were then sorted on a FACS
Vantage (Becton Dickinson) from the mouse BM and cultured in bulk or as
single cells in LTC and lymphomyeloid conditions. Ten control mice were
injected with the same number of mock-transduced cells.
Statistical analyses Statistical analyses were done using the Student t test.
Lentiviral vector-mediated gene transfer into CD34+ cells is enhanced by the central DNA flap First, using a 24-hour transduction protocol in the presence of cytokines, we compared the transduction efficiencies of CD34+ and CD34+CD38 CB cells with
HIV-1-derived vectors that lacked (HR18 vector) or
harbored (TRIP vector), the triple-stranded DNA flap structure (Figure
1A). In both vectors, EGFP transgene expression was driven from an internal CMV promoter. The presence of the HIV-1 DNA flap in
the lentiviral vector strikingly increased transduction efficiencies in
CD34+ CB cells, as measured by the percentage of
CD34+EGFP+ cells (Figure 1B, upper panel,
indicated by squares). This result was independent of the quantity of
vector used. In a total of 8 experiments, using a concentration of
vector particles corresponding to 500 ng P24/mL, 36% ± 16%
(mean ± SD; range, 17%-63%) and 11% ± 5% (range, 5%-20%) of
CD34+ cells expressed EGFP after exposure to the TRIP and
HR vectors, respectively (P = .0002). This higher
transduction efficiency of TRIP versus HR vector was also true for the
more immature CD34+CD38 cells because in 3 experiments using the same concentration of vector particles,
46% ± 28% (mean ± SD; range, 30%-78%) and only 11% ± 8%
(range, 5%-20%) of cells were EGFP+ after exposure to the
TRIP and HR vectors, respectively (Figure 1B, upper panel, indicated by
triangles). It is interesting to note that at high concentrations of
vector particles, the mean fluorescence intensity (MFI) was higher in
cells transduced with the TRIP vector compared to the HR vector (Figure
1B, middle panel), reflecting an increased level of EGFP protein in
TRIP-transduced cells. Combining both the percentage of
EGFP+ cells and the mean fluorescence intensity, the
presence of the DNA flap sequence could enhance EGFP synthesis in
CD34+ cells by more than a 10-fold factor (Figure 1B,
lower panel).
The central DNA flap promotes nuclear import of a lentiviral vector in CD34+ cells To determine whether the effect of the flap sequence on nuclear import was responsible for the observed increase in transduction efficiency, we precisely quantified the different forms of the intracellular vector DNA present within human CD34+ CB cells 48 hours after exposure to the TRIP or HR vectors (Figure 2). Once reverse-transcribed in the cytoplasm, the linear vector DNA is imported into the nucleus, where it either integrates or circularizes, generally into one LTR DNA circle. Using a specific strategy of digestion and probing (Figure 2A), these different DNA forms were accurately quantified by a Southern blot assay in CD34+-transduced cells. In cells exposed to the TRIP vector, 60% of the total vector DNA had integrated into the host cell DNA, 18% had circularized into one LTR circle, and 22% remained as stable unintegrated linear DNA. As previously described,28 2 LTR DNA circles were present only in trace amounts. Of note, this intracellular vector DNA profile was similar to the profile observed in cells infected with wild-type HIV-1,1 indicating an efficient nuclear import process. In cells exposed to the HR vector, a similar amount of vector DNA was synthesized, indicating that the presence of the DNA flap sequence did not influence the rate of reverse transcription. However, a marked alteration in the pattern of intracellular HR vector DNA was evident. In cells exposed to the HR vector, there was a striking accumulation of untranslocated linear DNA forms (representing up to 80% of total vector DNA), and only low amounts of nuclear forms (Figure 2B,C).
This intracellular DNA profile strongly suggests a defect in the
nuclear import of HR DNA, which leads to a decreased proportion of
nuclear viral DNA species and a concomitant increase in the proportion
of untranslocated linear DNA molecules. Thus, the strong defect in the
nuclear import of the HR vector DNA in CD34+ cells was
complemented by the insertion of the DNA flap sequence. Upon
establishing the benefit of the central DNA flap on gene transduction
in CD34+ and in CD34+CD38 The TRIP vector efficiently transduces CD34+ cells isolated from various sources We next tested the ability of the TRIP vector to transduce CD34+ cells from CB, PBMC, and BM cells. A TRIP vector inoculum corresponding to 100 ng P24/mL was used in all experiments. In 40 experiments, 40% ± 16% (range, 17%-78%) of CD34+ CB cells were transduced. Interestingly, omitting cytokines led to a drop in the transduction efficiency (9% ± 5%; range, 2%-19%; n = 14) as previously observed.27,31 We also successfully transduced CD34+ cells from adult sources such as cytokine-mobilized PBMC and BM cells (29% ± 10% and 41% ± 5%, respectively; n = 4) (Figure 3A). PCR analysis on CFC-derived colonies confirmed the proportion of progenitors that had been transduced (data not shown).
High-efficiency transduction of slow-dividing immature hematopoietic cells The main goal of gene transfer in human HSCs is to target immature stem cells with extensive proliferating and differentiating capacities. To demonstrate that the TRIP vector was able to transduce such cells, different approaches were used. First, we observed that both CD34+ and CD34+CD38 cells from
the same CB samples were susceptible to transduction by the TRIP vector
(mean transduction efficiencies of 41% ± 5% and 57% ± 6%,
respectively; n = 5) (Figures 1B and 3B). Second, as HSCs exhibit a
low mitotic activity,32 it was important to demonstrate
that slow-dividing or nondividing cells were transduced in our
protocol. To this end, CD34+ CB cells were labeled with
PKH26, a fluorescent dye that intercalates into the cell membrane
and is diluted as the cell divides. A homogeneous population of
PKH26bright CD34+ cells was sorted and
transduced. Immediately after exposure to the vector particles, more
than 95% of the CD34+ cells exhibited the same fluorescent
intensity as colcemid-treated cells, which indicated that they had not
undergone mitosis during the transduction period (Figure
4A, day 1). Indeed, 80% of these cells
had not yet begun to replicate their DNA (data not shown).
We then analyzed EGFP expression as a function of cell
division 48 hours after transduction as a function of cell division. As
it is generally believed that cells which are more immature divide
slowly in vitro in the presence of cytokines, we arbitrarily distinguished a "slow-dividing" (0 and 1 division illustrated in
Figure 4A, middle panel) cell population and a "rapid-dividing" (more than 2 divisions) cell population. Interestingly,
40.5% ± 17% of the slow-dividing cells and 36% ± 12% of the
rapid-dividing cells in these populations (n = 3) expressed EGFP,
respectively, indicating that they had been transduced at equivalent
levels (Figure 4A, right panel). To confirm the immaturity of the
slow-dividing cells, they were sorted (sorting gate shown in Figure 4A)
and tested for the presence of LTC-ICs and cells able to generate both
lymphoid (B and NK cells) and myeloid (granulomacrophagic cells)
progenies. The proportions of slow-dividing cells that were LTC-ICs
were 15% and 27% in experiments 1 and 2; of these LTC-ICs 33% and
50%, respectively, were EGFP+ by PCR analysis (Table 1).
In these same slow-dividing cells, lymphomyeloid progenitors were
identified. In experiment 1, 29.5% and in experiment 2, 24% of the
progenitors were multipotent, ie, capable of differentiation in B, NK,
and myeloid (M) cells. Of note, 40% of these clones also
differentiated into dendritic (D) cells (Figure 4B, experiment 1).
Importantly, 61% (experiment 1) and 37.5% (experiment 2) of these
multipotent lymphomyeloid progenitors were transduced (as assessed by
EGFP expression) compared to 22% to 45% of more mature
CD34+ cells that were committed to only one or 2 lineages
(Table 1).
Altogether these results indicate that the TRIP vector has the ability to transduce slow-dividing progenitors, which include primitive LTC-IC and multipotential (NK+B+M+D) progenitors. The lymphomyeloid potential of the slow-dividing sorted cells was similar to that observed with fresh CD34+ CB cells,19 indicating that contact with and/or transduction by the TRIP vector had no detrimental effect on the hematopoietic potential of CD34+ CB cells. Long-term NOD/SCID repopulating cells are transduced by the TRIP vector Finally, because long-term in vivo transplantability of human cells in NOD/SCID mice is considered a hallmark of cell immaturity,33 we injected CD34+ CB cells immediately after transduction into NOD/SCID mice (3 experiments). Fifteen weeks after transplantation, human hematopoietic cells (CD34+ progenitors, CD19+ B-lymphoid, and CD14/CD15+ granulocyte and myelomonocytic cells) were present in the BM of 10 of 11 mice. (The proportion of human cells varied between 0.5% and 62%, which is comparable to controls injected with untransduced cells). Surprisingly, in 2 experiments (6 mice), it was noted that whereas 50% of the injected cells expressed EGFP, more than 80% of the human cells detected 15 weeks later were EGFP+. Analysis of a representative mouse is shown in Figure 5A. To demonstrate that CD34+CD38 cells present in the BM of NOD/SCID
mice had been transduced and were functional, in one experiment these
cells were sorted and grown in lymphomyeloid bulk culture. After 2 weeks these cells produced CD19+ B, CD56+ NK,
and CD14+ monocytic cells and GPA+
erythroblasts in the presence of rhEPO, and all these cells expressed EGFP (Figure 5B). Moreover, analysis of the progeny of single-cell cultures indicated that multipotent EGFP+ B + NK + M
progenitors were present among CD34+CD38
human cells engrafted in NOD/SCID mice (Figure 5C) at a frequency of
20%, which is identical to that observed in comparable experiments with fresh CD34+ CB cells.19 In addition, this
cell population contained transduced LTC-ICs (data not shown). These
results definitively demonstrate that the TRIP vector efficiently
transduces functional primitive and multipotent
CD34+CD38 human CB cells with the ability to
sustain long-term hematopoiesis in NOD/SCID mice.
In an attempt to transduce very primitive human HSCs, we tested the efficiency of lentiviral vectors that in contrast to murine oncoretrovirus-derived vectors, can integrate in the genome of nondividing cells. Based on our recent demonstration that inclusion of the central DNA flap of HIV-1 dramatically enhances viral genome nuclear import,1 we derived a new construct, designated the TRIP vector, by inserting this central DNA flap in the HR vector. Here we show that this TRIP lentiviral vector, which encodes the EGFP transgene, can efficiently transduce very primitive human progenitors during a very short culture period (24 hours) in the presence of cytokines. This conclusion is based on 2 sets of data: (1) Approximately 40% of CD34+ cells could be transduced and could express the EGFP transgene during a sustained period of time. With selected batches of vector, it is of note that more than 60% of the CD34+ cells could reproducibly be transduced. (2) Similar high proportions of cells derived from myeloid LTC-ICs, multipotent lymphomyeloid progenitors, and long-term repopulating NOD/SCID mouse cells expressed EGFP, indicating the efficient transduction of progenitors functionally close to stem cells. A major determinant was the use of the modified vector
including the central DNA flap. Indeed, we first compared the
transduction efficiencies of HR and TRIP vectors on CD34+
and CD34+CD38 When a high concentration of TRIP vector particles was used for transduction, an increase in the fluorescence intensity of transduced cells was also observed. Because the insertion of the DNA flap sequence does not influence the transcriptional activity of the internal CMV promoter,1 the higher level of EGFP expression observed with the TRIP vector is likely to be due to multiple integration events of the vector genome into target cells. However, increasing the vector dose did not permit the CD34+ cell population to be transduced to homogeneity. This could be explained if a fraction of the CD34+ cell population was refractory to transduction by lentiviral vectors that, for instance, might be related to the variable proportion of cells being in the G0 or early G1 phase.34,35 In the case of HIV-1, it has been shown that in naive lymphocytes, which are in G0, the HIV replicative cycle aborts due to an incomplete reverse transcription process.36,37 A second possibility explaining the refractoriness of certain CD34+ cells to lentiviral vector transduction could be the lack of nuclear import of the retrotranscribed HIV-1 genome.38 Finally, toxicity is possibly induced at a high concentration of vector particles. The cPPT and CTS cis-active sequences, which are responsible for the formation of the DNA flap during reverse transcription, are located at the center of lentiviral genomes and overlap the 3'-region of the integrase coding sequence (POL). The crucial role of the DNA flap in vector DNA nuclear import most probably accounts for the previously reported heterogeneous efficiency of transduction of CD34+ cells by lentiviral vectors. Indeed, an efficient transduction is described using vectors in which the transgene replaces the envelope and/or Nef coding sequences of the HIV-1 genome.29,39,40 However, the HIV-1 GAG and POL proteins are encoded by such vector constructs, thereby representing a major disadvantage because deficient HIV particles are produced by the transduced cells. A much lower efficiency of transduction has been reported with classical "gutless" nonimmunogenic lentiviral vectors deleted for the entire coding sequences and thus for the central DNA flap sequence.27,41 In this study the HR vector appeared significantly less efficient than
the TRIP vector in transducing CD34+ and
CD34+CD38 Previous experiments have shown that NOD/SCID-repopulating cells could
be transduced by the HR vector, with transduction of as many as 27% of
human CD45+ cells.41 As we expected, the TRIP
vector resulted in a better transduction efficiency in
NOD/SCID-repopulating cells. Surprisingly, whereas only 50% of
TRIP-transduced CD34+ CB cells expressed EGFP 48 hours
after transduction in 2 experiments, at least 80% of their progeny
harvested from BM of engrafted NOD/SCID mice were transduced (6 of 7 recipient mice). Similarly, Miyoshi et al41 detected a
transduction efficiency of 35% in CFCs recovered from NOD/SCID mice
even though the original clonogenic progenitors were transduced with an
efficiency of 12% to 17%. These data have led us to speculate that
the NOD/SCID-repopulating cells and multipotent progenitors are
particularly susceptible to transduction by lentiviral vectors.
Moreover, during 2 in vitro experiments, single progenitors able to
generate a progeny of B, NK, and M cells appeared more efficiently
transduced than monopotent and bipotent progenitors (Table 1), and
CD34+CD38 We demonstrate here that the flap structure restores the nuclear import defect of HIV-1-based replacement vector genomes, thereby dramatically increasing the transduction efficiencies of human CD34+ hematopoietic populations from different sources. TRIP, the flap-containing vector, is able to efficiently transduce human HSCs defined by their slow-dividing rate, multipotentiality, and ability to establish long-term active human hematopoiesis in the BM of NOD/SCID mice. Importantly, the transduction process of CD34+ cells did not alter their quantitative and qualitative differentiation potentials. The highly efficient transduction of human HSCs by the TRIP vector will undoubtedly open multiple gene therapy applications and help to resolve fundamental questions regarding the biology of human HSCs.
We would like to thank A. Rouchès and P. Ardouin for their expertise in the production of NOD/SCID mice, V. Schiavon for cell sorting, B. Izac for technical assistance, Dr Van Nifderick and colleagues for kindly providing CB samples, and Naomi Taylor for critical comments on the manuscript. We also thank Amgen for rhSCF, Kirin for rhPEG-MGDF, CILAG for rhEPO, and Immunex for rhFlt3-L.
Submitted April 12, 2000; accepted July 30, 2000.
A.S. and F.P. contributed equally to this work.
Supported by grant ACC-SV 96-335 from Ministère de la Recherche et de la Technologie, Paris; grants 1137 and 9225 from the Association de la Recherche contre le Cancer, Villejuif; and grants from the Institut National de la Santé et de la Recherche Médicale, Paris; from the Institut Gustave Roussy, Villejuif; from the Agence Nationale de Recherche contre le Sida, Paris; from the Association Française contre les Myopathies, Paris; and Electricité de France, Paris, France.
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: Anne Dubart-Kupperschmitt, INSERM U474, Maternité de Port Royal, 123 Bd de Port Royal, 75014 Paris, France; e-mail: dubart{at}cochin.inserm.fr; or Pierre Charneau, Unité d'Oncologie Virale, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France; e-mail: charneau{at}pasteur.fr.
1. Zennou V, Guetard D, Petit C, Nerhbass U, Montagnier L, Charneau P. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell. 2000;101:1-20[Medline] [Order article via Infotrieve].
2.
Weinberg JB, Matthews TJ, Cullen BR, Malim MH.
Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.
J Exp Med.
1991;174:1477-1482
3.
Bukrinsky MI, Sharova N, Dempsey MP, et al.
Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.
Proc Natl Acad Sci U S A.
1992;89:6580-6584 4. Bukrinsky MI, Haggerty S, Dempsey MP, et al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells [see comments]. Nature. 1993;365:666-669[Medline] [Order article via Infotrieve]. 5. Emerman M, Bukrinski M, Stevenson M. HIV-1 infection of non-dividing cells: reply to Freed ED and Martin MA. Nature. 1994;369:108. 6. Popov S, Rexach M, Zybarth G, et al. Viral protein R regulates nuclear import of the HIV-1 pre-integration complex. EMBO J. 1998;17:909-917[Medline] [Order article via Infotrieve].
7.
von Schwedler U, Kornbluth RS, Trono D.
The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes.
Proc Natl Acad Sci U S A.
1994;91:6992-6996
8.
Gallay P, Hope T, Chin D, Trono D.
HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway.
Proc Natl Acad Sci U S A.
1997;94:9825-9830 9. Fouchier RA, Meyer BE, Simon JH, Fischer U, Malim MH. HIV-1 infection of non-dividing cells: evidence that the amino-terminal basic region of the viral matrix protein is important for Gag processing but not for post-entry nuclear import. EMBO J. 1997;16:4531-4539[Medline] [Order article via Infotrieve]. 10. Freed EO, Martin MA. HIV-1 infection of non-dividing cells [letter; see comment]. Nature. 1994;369:107-108[Medline] [Order article via Infotrieve]. 11. Freed EO, Englund G, Martin MA. Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection. J Virol. 1995;69:3949-3954[Abstract]. 12. Freed EO, Englund G, Maldarelli F, Martin MA. Phosphorylation of residue 131 of HIV-1 matrix is not required for macrophage infection. Cell. 1997;88:171-173[Medline] [Order article via Infotrieve].
13.
Charneau P, Clavel F.
A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract.
J Virol.
1991;65:2415-2421
14.
Charneau P, Alizon M, Clavel F.
A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication.
J Virol.
1992;66:2814-2820 15. Charneau P, Mirambeau G, Roux P, Paulous S, Buc H, Clavel F. HIV-1 reverse transcription: a termination step at the center of the genome. J Mol Biol. 1994;241:651-662[Medline] [Order article via Infotrieve]. 16. Lavigne M, Roux P, Buc H, Schaeffer F. DNA curvature controls termination of plus strand DNA synthesis at the centre of HIV-1 genome. J Mol Biol. 1997;266:507-524[Medline] [Order article via Infotrieve]. 17. Miller AD. Development and application of retroviral vectors. In: Coffin JM,Hughes SH,Varmus HE, eds. Retroviruses. Cold Spring, NY: Cold Spring Harbor Laboratory Press; 1997:437-473. 18. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [see comments]. Science. 1996;272:263-267[Abstract].
19.
Robin C, Pflumio F, Vainchenker W, Coulombel L.
Identification of lymphomyeloid primitive progenitor cells in fresh human cord blood and in the marrow of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice transplanted with human CD34(+) cord blood cells.
J Exp Med.
1999;189:1601-1610 20. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol. 1997;15:871-875[Medline] [Order article via Infotrieve].
21.
Zufferey R, Dull T, Mandel RJ, et al.
Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.
J Virol.
1998;72:9873-9880
22.
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
23.
Debili N, Wendling F, Katz A, et al.
The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors.
Blood.
1995;86:2516-2525
24.
Croisille L, Auffray I, Katz A, Izac B, Vainchenker W, Coulombel L.
Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38
25.
Issaad C, Croisille L, Katz A, Vainchenker W, Coulombel L.
A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38[minud] progenitor cells in long-term cultures and semisolid assays.
Blood.
1993;81:2916-2924 26. Marandin A, Dubart A, Pflumio F, et al. Retrovirus-mediated gene transfer into human CD34+38low primitive cells capable of reconstituting long-term cultures in vitro and nonobese diabetic-severe combined immunodeficiency mice in vivo. Hum Gene Ther. 1998;9:1497-1511[Medline] [Order article via Infotrieve].
27.
Case SS, Price MA, Jordan CT, et al.
Stable transduction of quiescent CD34(+)CD38( 28. Barbosa P, Charneau P, Dumey N, Clavel F. Kinetic analysis of HIV-1 early replicative steps in a coculture system. AIDS Res Hum Retroviruses. 1994;10:53-59[Medline] [Order article via Infotrieve].
29.
Huang S, Terstappen LW.
Lymphoid and myeloid differentiation of single human CD34+, HLA-DR+, CD38
30.
Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM.
A functional comparison of CD34 + CD38
31.
Sutton RE, Reitsma MJ, Uchida N, Brown PO.
Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent.
J Virol.
1999;73:3649-3660
32.
Gothot A, van der Loo JC, Clapp DW, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese diabetic/severe combined immune-deficient mice.
Blood.
1998;92:2641-2649 33. Dick JE, Bhatia M, Gan O, Kapp U, Wang JC. Assay of human stem cells by repopulation of NOD/SCID mice. Stem Cells. 1997;15:199-203.
34.
Sutton RE, Wu HT, Rigg R, Bohnlein E, Brown PO.
Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells.
J Virol.
1998;72:5781-5788
35.
Korin YD, Zack JA.
Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells.
J Virol.
1998;72:3161-3168 36. Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990;61:213-222[Medline] [Order article via Infotrieve]. 37. Tang S, Patterson B, Levy JA. Highly purified quiescent human peripheral blood CD4+ T cells are infectible by human immunodeficiency virus but do not release virus after activation. J Virol. 1995;69:5659-5665[Abstract]. 38. Stevenson M, Stanwick TL, Dempsey MP, Lamonica CA. HIV-1 replication is controlled at the level of T cell activation and proviral integration. EMBO J. 1990;9:1551-1560[Medline] [Order article via Infotrieve].
39.
Reiser J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M.
Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles.
Proc Natl Acad Sci U S A.
1996;93:15266-15271
40.
Uchida N, Sutton RE, Friera AM, et al.
HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells.
Proc Natl Acad Sci U S A.
1998;95:11939-11944
41.
Miyoshi H, Smith KA, Mosier DE, Verma IM, Torbett BE.
Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.
Science.
1999;283:682-686
42.
Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT.
Functional isolation and characterization of human hematopoietic stem cells.
Science.
1995;267:104-108
© 2000 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  L. Wang, D. Li, T. A. Dawson, and D. J. Paterson Long-term effect of neuronal nitric oxide synthase over-expression on cardiac neurotransmission mediated by a lentiviral vector J. Physiol., July 15, 2009; 587(14): 3629 - 3637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Armstrong, P. B. de la Grange, B. Gerby, M.-C. Rouyez, J. Calvo, M. Fontenay, N. Boissel, H. Dombret, A. Baruchel, J. Landman-Parker, et al. NOTCH is a key regulator of human T-cell acute leukemia initiating cell activity Blood, February 19, 2009; 113(8): 1730 - 1740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Alonso, I. Ortega-Perez, M. S. Grubb, J.-P. Bourgeois, P. Charneau, and P.-M. Lledo Turning Astrocytes from the Rostral Migratory Stream into Neurons: A Role for the Olfactory Sensory Organ J. Neurosci., October 22, 2008; 28(43): 11089 - 11102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Ao, G. Huang, H. Yao, Z. Xu, M. Labine, A. W. Cochrane, and X. Yao Interaction of Human Immunodeficiency Virus Type 1 Integrase with Cellular Nuclear Import Receptor Importin 7 and Its Impact on Viral Replication J. Biol. Chem., May 4, 2007; 282(18): 13456 - 13467. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Thomas, Y. A. Voronin, G. N. Nikolenko, J. Chen, W.-S. Hu, and V. K. Pathak Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis J. Virol., May 1, 2007; 81(9): 4798 - 4807. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ly, Y. Kawase, R. Yoneyama, and R. J. Hajjar Gene Therapy in the Treatment of Heart Failure Physiology, April 1, 2007; 22(2): 81 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Garfinkel, K. M. Stefanisko, K. M. Nyswaner, S. P. Moore, J. Oh, and S. H. Hughes Retrotransposon Suicide: Formation of Ty1 Circles and Autointegration via a Central DNA Flap J. Virol., December 15, 2006; 80(24): 11920 - 11934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Arhel, S. Munier, P. Souque, K. Mollier, and P. Charneau Nuclear import defect of human immunodeficiency virus type 1 DNA flap mutants is not dependent on the viral strain or target cell type. J. Virol., October 1, 2006; 80(20): 10262 - 10269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wurtzer, A. Goubard, F. Mammano, S. Saragosti, D. Lecossier, A. J. Hance, and F. Clavel Functional Central Polypurine Tract Provides Downstream Protection of the Human Immunodeficiency Virus Type 1 Genome from Editing by APOBEC3G and APOBEC3B. J. Virol., April 1, 2006; 80(7): 3679 - 3683. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Van Damme, L. Thorrez, L. Ma, H. Vandenburgh, J. Eyckmans, F. Dell'Accio, C. De Bari, F. Luyten, D. Lillicrap, D. Collen, et al. Efficient Lentiviral Transduction and Improved Engraftment of Human Bone Marrow Mesenchymal Cells Stem Cells, April 1, 2006; 24(4): 896 - 907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. MORDELET, K. KISSA, A. CRESSANT, F. GRAY, S. OZDEN, C. VIDAL, P. CHARNEAU, and S. GRANON Histopathological and cognitive defects induced by Nef in the brain FASEB J, December 1, 2004; 18(15): 1851 - 1861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Soda, K. Tani, Y. Bai, M. Saiki, M. Chen, K. Izawa, S. Kobayashi, S. Takahashi, K. Uchimaru, T. Kuwabara, et al. A novel maxizyme vector targeting a bcr-abl fusion gene induced specific cell death in Philadelphia chromosome-positive acute lymphoblastic leukemia Blood, July 15, 2004; 104(2): 356 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Davis, L. Humeau, V. Slepushkin, G. Binder, L. Korshalla, Y. Ni, E. O. Ogunjimi, L.-F. Chang, X. Lu, and B. Dropulic ABC transporter inhibitors that are substrates enhance lentiviral vector transduction into primitive hematopoietic progenitor cells Blood, July 15, 2004; 104(2): 364 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. A. Voronin and V. K. Pathak Frequent Dual Initiation in Human Immunodeficiency Virus-Based Vectors Containing Two Primer-Binding Sites: a Quantitative In Vivo Assay for Function of Initiation Complexes J. Virol., May 15, 2004; 78(10): 5402 - 5413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Ravet, D. Reynaud, M. Titeux, B. Izac, S. Fichelson, P.-H. Romeo, A. Dubart-Kupperschmitt, and F. Pflumio Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesis Blood, May 1, 2004; 103(9): 3326 - 3335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Ao, X. Yao, and E. A. Cohen Assessment of the Role of the Central DNA Flap in Human Immunodeficiency Virus Type 1 Replication by Using a Single-Cycle Replication System J. Virol., March 15, 2004; 78(6): 3170 - 3177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Goujon, L. Jarrosson-Wuilleme, J. Bernaud, D. Rigal, J.-L. Darlix, and A. Cimarelli Heterologous Human Immunodeficiency Virus Type 1 Lentiviral Vectors Packaging a Simian Immunodeficiency Virus-Derived Genome Display a Specific Postentry Transduction Defect in Dendritic Cells J. Virol., September 1, 2003; 77(17): 9295 - 9304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Segovia, G. Guenechea, J. M. Gallego, J. M. Almendral, and J. A. Bueren Parvovirus Infection Suppresses Long-Term Repopulating Hematopoietic Stem Cells J. Virol., August 1, 2003; 77(15): 8495 - 8503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. V. Parry, C. A. Rumbley, L. H. Vandenberghe, C. H. June, and J. L. Riley CD28 and Inducible Costimulatory Protein Src Homology 2 Binding Domains Show Distinct Regulation of Phosphatidylinositol 3-Kinase, Bcl-xL, and IL-2 Expression in Primary Human CD4 T Lymphocytes J. Immunol., July 1, 2003; 171(1): 166 - 174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Piacibello, S. Bruno, F. Sanavio, S. Droetto, M. Gunetti, L. Ailles, F. S. de Sio, A. Viale, L. Gammaitoni, A. Lombardo, et al. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice Blood, December 15, 2002; 100(13): 4391 - 4400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lyonnais, C. Hounsou, M.-P. Teulade-Fichou, J. Jeusset, E. L. Cam, and G. Mirambeau G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome Nucleic Acids Res., December 1, 2002; 30(23): 5276 - 5283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Limon, N. Nakajima, R. Lu, H. Z. Ghory, and A. Engelman Wild-Type Levels of Nuclear Localization and Human Immunodeficiency Virus Type 1 Replication in the Absence of the Central DNA Flap J. Virol., October 25, 2002; 76(23): 12078 - 12086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Dvorin, P. Bell, G. G. Maul, M. Yamashita, M. Emerman, and M. H. Malim Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import J. Virol., October 25, 2002; 76(23): 12087 - 12096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Hamaguchi, A. Ooka, A. Brun, J. Richter, N. Dahl, and S. Karlsson Gene transfer improves erythroid development in ribosomal protein S19-deficient Diamond-Blackfan anemia Blood, September 26, 2002; 100(8): 2724 - 2731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Ducrey-Rundquist, M. Guyader, and D. Trono Modalities of Interleukin-7-Induced Human Immunodeficiency Virus Permissiveness in Quiescent T Lymphocytes J. Virol., August 12, 2002; 76(18): 9103 - 9111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. VandenDriessche, L. Thorrez, L. Naldini, A. Follenzi, L. Moons, Z. Berneman, D. Collen, and M. K. L. Chuah Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo Blood, July 18, 2002; 100(3): 813 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Srinivasakumar, M. Zaboikin, T. Zaboikina, and F. Schuening Evaluation of Tat-Encoding Bicistronic Human Immunodeficiency Virus Type 1 Gene Transfer Vectors in Primary Canine Bone Marrow Mononuclear Cells J. Virol., June 14, 2002; 76(14): 7334 - 7342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Maurice, E. Verhoeyen, P. Salmon, D. Trono, S. J. Russell, and F.-L. Cosset Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide Blood, April 1, 2002; 99(7): 2342 - 2350. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pfeifer, M. Ikawa, Y. Dayn, and I. M. Verma Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos PNAS, February 19, 2002; 99(4): 2140 - 2145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Scherr, K. Battmer, U. Blomer, B. Schiedlmeier, A. Ganser, M. Grez, and M. Eder Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells Blood, January 15, 2002; 99(2): 709 - 712. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
,
) and HR (
, 
) vectors. CD34+ CB cells (
100) during 24 hours. Cells were then washed and cultured in vitro in conditions that
support lymphoid and myeloid differentiation
long-term culture-initiating cells.
Blood.
1994;84:4116-4124