Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver

Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.     

A pilot study comparing severely and persistently mentally ill opiate-addicted patients in dual-diagnosis treatment with patients in methadone maintenance

Retroviral vector gene transfer strategies are currently being developed to treat a variety of hematopoietic disorders. To date, genetic modification of human pluripotent hematopoietic stem cells has been inefficient. In the present study we developed reagents and procedures for rapidly screening retroviral vector gene transfer conditions using a multiparameter fluorescence-activated cell sorting (FACS) assay. To identify transduced cells using FACS analysis, we developed a retroviral vector, termed MN, which stably expressed high levels of a truncated version of the low-affinity nerve growth factor receptor (LNGFR). In addition, procedures were developed for enriching CD34+ cells from cryopreserved umbilical cord blood. These cells were transduced with MN and evaluated using multiparameter FACS analysis for expression of CD34, CD38, and LNGFR. Stem cell maintenance was determined by measuring the CD34hi and CD34hiCD38lo/- cells remaining after ex vivo gene transfer. Gene transfer into these cells was measured by evaluating cells expressing high levels of LNGFR. Initial studies with this assay and with in vitro functional assays indicated that retroviral gene transfer following pre-incubation with a variety of cytokines in serum containing conditions resulted in 1) poor maintenance of hematopoietic stem cells and 2) gene transfer predominantly in relatively mature cells. When gene transfer in serum-free conditions was performed, some improvement was observed in the maintenance of cells retaining primitive immunophenotypes with no reduction in the gene transfer efficiency. The MN vector and multiparameter FACS analysis will be useful in efficiently screening ongoing efforts designed to improve stem cell gene transfer.     

Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.     

Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.     

Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.     

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.     

Development of novel cell surface CD34-targeted recombinant adenoassociated virus vectors for gene therapy

Recombinant adenoassociated virus (rAAV) type 2 vectors have been used to transduce a wide variety of cell types, including hematopoietic progenitor cells. For in vivo gene transfer, it is desirable to have an rAAV vector that specifically transduces selected target cells. As a first step toward generating an rAAV vector capable of targeting delivery in vivo, we have engineered a chimeric protein combining the AAV capsid protein and the variable region of a single-chain antibody against human CD34 molecules, a cell surface marker for hematopoietic stem/progenitor cells. Inclusion of the chimeric CD34 single-chain antibody-AAV capsid proteins within an rAAV virion significantly increased the preferential infectivity of rAAV for the CD34+ human myoleukemia cell line KG-1, which is normally refractory to rAAV transduction. Antibodies against the single-chain antibody and the CD34 protein blocked this transduction. This chimeric vector represents a significant improvement in the host range of rAAV and the first step toward specific gene delivery by rAAV vectors to cells of choice, in this case, hematopoietic progenitor cells, for the treatment of human disease.     

Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.     

High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.     

Retrovirus-mediated transfer of the human alpha-L-iduronidase cDNA into human hematopoietic progenitor cells leads to correction in trans of Hurler fibroblasts

Hurler syndrome (mucopolysaccharidosis IH or MPS IH) is a congenital mucopolysaccharide storage disorder resulting from a genetic deficiency of alpha-L-iduronidase (IDUA), which is required for lysosomal degradation of glycosaminoglycans heparan sulfate and dermatan sulfate. Even though histocompatible bone marrow transplantation has been applied for the treatment of Hurler syndrome, gene therapy via autologous bone marrow transplantation (BMT) may be more beneficial for this disease. Two retroviral vectors containing a full-length human IDUA cDNA were constructed using Moloney murine leukemia virus (MoMLV)-based vector backbones. High-titer vector-producing clones containing the L-HuID-SN and MFG-HuID retroviral vectors were established. The efficiency of gene transfer into primitive human CD34+ hematopoietic cells using both retroviral vectors is in the range of 18-23%. The level of enzyme expression in transduced primary bone marrow cells was increased 40- to 50-fold compared with that of sham-transduced cells. Enzyme produced by the progeny of the transduced human CD34+ cells carrying IDUA cDNA corrected Hurler fibroblasts via mannose-6-phosphate receptors. These findings suggest that genetically modified hematopoietic progenitor cells can potentially be useful for gene therapy of Hurler syndrome.     

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.     

Ex vivo culture of CD34+/Lin-/DR- cells in stroma-derived soluble factors, interleukin-3, and macrophage inflammatory protein-1alpha maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1alpha (MIP-1alpha). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin-/DR- cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin-/DR- cells assayed in SNC cultures supplemented with IL-3 and MIP-1alpha. When CD34+/Lin-/DR- progeny from the SNC culture were plated sequentially into "NK cell progenitor switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3- NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1alpha directly to "NK cell differentiation" conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33- cells present after SNC cultures with IL-3 and MIP-1alpha, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33- population similar to fresh sorted CD34+/Lin-/DR- cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1alpha, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.     

Abrogation of TGF-beta activity during retroviral transduction improves murine hematopoietic progenitor and repopulating cell gene transfer efficiency

Transforming growth factor-beta has complex activities on hematopoietic cells. We have previously shown that murine long-term repopulating activity is compromised by ex vivo culture in TGF-beta 1 and conversely is increased by abrogating endogenous TGF-beta activity with a neutralizing antibody. In the current study, we investigated the effect of abrogation of autocrine or paracrine TGF-beta present during retroviral transduction on gene transfer efficiency to primitive hematopoietic cells. Murine marrow cells were cultured and retrovirally transduced for 4 days in the presence of interleukin-3, interleukin-6 and stem cell factor, and either a neutralizing anti-TGF-beta antibody or an isotype control. Committed progenitor cells were analyzed for gene transfer efficiency, and cells were also injected into W/Wv recipient mice for analysis of transduction of long-term repopulating cells. The progenitor (CFU-C) transduction efficiency in the presence of anti-TGF-beta was significantly greater. Semiquantitative PCR analysis and Southern blot analysis for the retroviral marker gene in the blood and bone marrow of recipient mice revealed a significant increase in the transduction efficiency of long-term repopulating cells after culture and transduction in the presence of the anti-TGF-beta. Thus neutralization of TGF-beta activity during retroviral transduction allows more efficient gene transfer into primitive murine hematopoietic cells and may prove beneficial in future clinical gene transfer or therapy trials.     

CD164, a novel sialomucin on CD34(+) and erythroid subsets, is located on human chromosome 6q21

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34(+) hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34(+) cells to bone marrow stroma and by negatively regulating CD34(+) hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34(+) cells are CD164(+), with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34(+) human bone marrow and cord blood cells that were CD38(lo/-) or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34(+)CD71(lo/-) or CD34(+)CD33(lo/-) cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34(+)CD164(103B2/9E10)lo/- cells in bone marrow are mainly CD19(+) B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34(lo/-)CD19(+) and CD34(lo/-)CD20(+) B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34(lo/-)CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21.     

Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.     

The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis

Mucin-like molecules represent an emerging family of cell surface glycoproteins expressed by cells of the hematopoietic system. We report the isolation of a cDNA clone that encodes a novel transmembrane isoform of the mucin-like glycoprotein MGC-24, expressed by both hematopoietic progenitor cells and elements of the bone marrow (BM) stroma. This molecule was clustered as CD164 at the recent workshop on human leukocyte differentiation antigens. CD164 was identified using a retroviral expression cloning strategy and two novel monoclonal antibody (MoAb) reagents, 103B2/9E10 and 105.A5. Both antibodies detected CD164/MGC-24v protein expression by BM stroma and subpopulations of the CD34(+) cells, which include the majority of clonogenic myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (blast-forming unit-erythroid [BFU-E]) progenitors and the hierarchically more primitive precursors (pre-CFU). Biochemical and functional characterization of CD164 showed that this protein represents a homodimeric molecule of approximately 160 kD. Functional studies demonstrate a role for CD164 in the adhesion of hematopoietic progenitor cells to BM stromal cells in vitro. Moreover, antibody ligation of CD164 on primitive hematopoietic progenitor cells characterized by the cell surface phenotype CD34(BRIGHT)CD38(-) results in the decreased recruitment of these cells into cell cycle, suggesting that CD164 represents a potent signaling molecule with the capacity to suppress hematopoietic cell proliferation.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.     

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.