Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.

Haematopoietic stem cells in umbilical cord blood are an attractive target for gene therapy of inborn errors of metabolism. Three neonates with severe combined immunodeficiency were treated by retroviral-mediated transduction of the CD34+ cells from their umbilical cord blood with a normal human adenosine deaminase complementary DNA followed by autologous transplantation. The continued presence and expression of the introduced gene in leukocytes from bone marrow and peripheral blood for 18 months demonstrates that umbilical cord blood cells may be genetically modified with retroviral vectors and engrafted in neonates for gene therapy.

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates.

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene.

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

Lentiviral vectors with amplified beta cell-specific gene expression.

An important goal of gene therapy is to be able to deliver genes, so that they express in a pattern that recapitulates the expression of an endogenous cellular gene. Although tissue-specific promoters confer selectivity, in a vector-based system, their activity may be too weak to mediate detectable levels in gene-expression studies. We have used a two-step transcriptional amplification system to amplify gene expression from lentiviral vectors using the human insulin promoter. In this system, the human insulin promoter drives expression of a potent synthetic transcription activator (the yeast GAL4 DNA-binding domain fused to the activation domain of the Herpes simplex virus-1 VP16 activator), which in turn activates a GAL4-responsive promoter, driving the enhanced green fluorescent protein reporter gene. Vectors carrying the human insulin promoter did not express in non-beta-cell lines, but expressed in murine insulinoma cell lines, indicating that the human insulin promoter was capable of conferring cell specificity of expression. The insulin-amplifiable vector was able to amplify gene expression five to nine times over a standard insulin-promoter vector. In primary human islets, gene expression from the insulin-promoted vectors was coincident with insulin staining. These vectors will be useful in gene-expression studies that require a detectable signal and tissue specificity.

Tissue-specific restriction of cyclophilin A-independent HIV-1- and SIV-derived lentiviral vectors.

The host factor alpha isoform of the tripartite motif 5 (TRIM5alpha) restricts human immunodeficiency virus type 1 (HIV-1) infection in certain non-human primate species. Restriction of HIV-1 is enhanced by binding of the viral capsid to cyclophilin A (CypA) in target cells, although CypA is not absolutely required for restriction in rhesus macaque cells. Simian immunodeficiency virus (SIV) is not restricted by rhesus macaque TRIM5alpha and its capsid does not bind to CypA. Here, the effect of lentiviral CypA dependence on restriction in different tissues was examined by engineering an HIV-1 capsid quadruple mutant (V(86)P/H(87)Q/I(91)V/M(96)I) lentiviral vector (HIV(quad)) that is CypA-independent. Whereas HIV-1 was restricted in rhesus macaque and owl monkey epithelial cells, infection with the HIV(quad) vector was efficient at high viral concentrations. In contrast, HIV(quad) was largely restricted in primary rhesus macaque CD34(+) cells. Human epithelial and primary CD34(+) cells were permissive for HIV-1, HIV(quad) and SIV, whereas transduction of human T cells by HIV(quad) or SIV was impaired. The restrictive human cells did not express increased levels of TRIM5alpha, and restriction was not relieved by abolishing CypA, consistent with HIV(quad) and SIV being CypA-independent. Pseudotyping of lentiviral vectors with the gibbon ape leukemia virus envelope altered their sensitivity to perturbations of the virus-CypA interaction compared to pseudotyping with vesicular stomatitis virus glycoproteins, suggesting that the viral entry pathway modulates restriction. Together, these studies reveal that an HIV-1 capsid quadruple mutant can partially overcome lentiviral restriction in non-human primate epithelial cells, but not in hematopoietic cells. Similarly, human cells vary in their permissiveness for CypA-independent lentiviruses, and suggest the presence of tissue-specific factor(s) that can inhibit lentiviral transduction independently of viral interaction with TRIM5alpha and CypA.

Gene therapy for childhood immunological diseases.

Gene therapy using autologous hematopoietic stem cells (HSC) that are corrected with the normal gene may have a beneficial effect on blood cell production or function, without the immunologic complications of allogeneic HSC transplantation. Childhood immunological diseases are highly favorable candidates for responses to gene therapy using HSC. Hemoglobinopathies, lysosomal and metabolic disorders and defects of hematopoietic stem and progenitor cells should also be ameliorated by gene therapy using autologous HSC. At present, gene therapy has been beneficial for patients with XSCID, ADA-deficient SCID and chronic granulomatous disease. The principle that partial marrow conditioning increases engraftment of gene-corrected HSC has been demonstrated. Clinical trials are being developed in Europe and the United States to treat several other genetic blood cell disorders. This progress is tempered by the serious complication observed in XSCID patients developing T lymphoproliferative disease. New methods for gene transfer (lentiviral and foamy viral vectors, semi-viral systems and gene correction) may retain or further increase the efficacy and decrease the risks from gene therapy using HSC. Ultimately, the relative benefits and risks of autologous gene therapy will be weighed against other available options (for example, allogeneic HSCT) to determine the treatment of choice.

Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity.

Overexpression of tissue inhibitor of metalloproteinases-2 retroviral-mediated gene transfer in vivo inhibits tumor growth and invasion.

We have demonstrated previously that overexpression of tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of matrix-degrading metalloproteinases, not only inhibits the invasive and metastatic behavior of tumor cells but also significantly decreases tumor growth in vivo (Y. A. DeClerck et at, Cancer Res., 52: 701-708, 1992). This latter effect was found to be dependent on the ability of TIMP-2 to prevent the degradation of the collagen matrix (A. M. Montgomery et al., Cancer Res., 54: 5467-5473, 1994). In this report, we have overexpressed TIMP-2 in tumor tissue by retroviral-mediated gene transfer into tumor cells by co-injecting s.c. in nude mice tumorigenic c-Ha-ras-transfected rat embryo fibroblasts with irradiated packaging cells producing high titer retroviral vectors containing the human TIMP-2 cDNA. The growth rate of tumors derived from cells co-injected with the TIMP-2 vector producer cells was significantly slower than the growth rate of tumors derived from cells co-injected with packaging cells producing a retrovirus containing the Escherichia coli beta-galactosidase gene. The transduction efficiency was estimated at 13%, and the production of a functional human TIMP-2 in tumor cells transduced with the TIMP-2-containing vector was documented. Furthermore, histological analysis of tumors derived from tumor cells co-injected with the TIMP-2 vector producer cells revealed the presence of a thick connective tissue capsule and a lack of local invasion. The data indicate that retroviral-mediated transduction of TIMP-2 cDNA into a limited population of tumor cells in vivo is sufficient to increase the accumulation of connective tissue proteins in tumor tissue, to inhibit growth, and to prevent local invasion.

Immunotherapy against murine leukemia.

The central hypothesis underlying specific anti-leukemia immunotherapy is that leukemic cells express antigenic determinants not expressed on their counterpart normal adult cells. We have developed a murine myeloid leukemia/tumor immunization model using the low-immunogenic WEHI3 leukemia in syngeneic mice. Mice preimmunized with irradiated, transduced IL-7-producing WEHI3 cells showed systemic protection and rejection of a lethal dose of intravenously (i.v.) injected parental WEHI3 cells (5 x 10(4)) with 40% long-term survival. When vaccinated with a mixture of parental WEHI3 cells and IL-2-producing NIH-3T3 fibroblasts (5 x 10(5)), 60% survival was observed. Vaccination with murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing WEHI3 cells resulted in only 20% survival of i.v. challenged mice, and the additional combination of IL-2- and IL-7-producing vaccine did not reveal any additive or synergistic effects. Immunizing mice with a pre-established leukemia burden (injected with 5 x 10(4) WEHI3 cells, i.v., 3 days prior to immunization) did not cure or result in a prolongation of survival, indicating that improved methods of immunization are needed. Taken together, we have identified IL-7 and IL-2 as effective cytokines in our leukemia/vaccination model with only marginal activity by GM-CSF.

Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells.

In the mouse mammary tumor virus (MMTV)-infected mouse T-lymphoma cell line W7MG1, glucocorticoid hormone regulates two aspects of MMTV gene expression: hormone stimulates MMTV gene transcription and increases the ratio of mature envelope proteins to envelope precursor protein produced. To separate these two effects and determine the mechanism by which hormone regulates the conversion of the envelope precursor Pr74 to the mature cleaved products gp52 and gp33, we constructed expression vectors in which the envelope gene is constitutively transcribed. Surprisingly, the envelope precursor protein Pr74 encoded by two independently isolated, allelic envelope genes behaved differently. Pr74-P (encoded by the ENV/P gene) was processed efficiently to the mature products gp52 and gp33, independently of the level of expression, hormonal induction of cellular genes, or the presence of other MMTV proteins. In contrast, under the same conditions, Pr74-N (encoded by the ENV/N gene) was not processed further despite being relatively stable. In sucrose gradient analyses, Pr74-P sedimented as monomers, whereas Pr74-N was found in high mol wt aggregates of heterogeneous size. Coimmunoprecipitation analysis determined that Pr74-N associated with BiP, whereas Pr74-P did not. This is indicative of improper folding of Pr74-N in the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral vector-mediated gene transfer into primitive human hematopoietic progenitor cells: effects of mast cell growth factor (MGF) combined with other cytokines.

Retroviral vector-mediated gene transfer into human hematopoietic stem cells may permit gene therapy of numerous genetic diseases. Stimulation of marrow with hematopoietic growth factors (HGFs) has been shown to increase the level of retroviral transduction. We have examined the effects of recombinant human mast cell growth factor (MGF), alone and in combination with other HGFs, on the efficiency of gene transfer into human hematopoietic progenitor cells. MGF acts in concert with interleukin 3 (IL-3) and interleukin 6 (IL-6) to increase the percentage of CD34+ progenitors transduced with a retroviral vector expressing the neo gene. The most potent combination of growth factors that we examined, interleukin 1 (IL-1)/IL-3/IL-6/MGF, resulted in the conferral of G418 resistance to 45% of progenitors and long-term culture-initiating cells. Extending the time of cocultivation of the marrow cells with the vector-producing cells did not further increase gene transfer frequency, suggesting that the amount of available vector is not limiting. To analyze the effects of the HGF on gene transfer into more primitive hematopoietic progenitors, CD34+ cells were isolated from marrow samples that were purged of committed progenitor cells by treatment with 4-hydroperoxycyclophosphamide (4-HC). Preculturing the CD34+ 4-HC-treated cells with the combination of four HGF (IL-1/IL-3/IL-6/MGF) permitted transduction of 20%-28% of the progenitors that formed colonies after 30 days in culture. These results demonstrate that MGF in combination with other HGFs enhances gene transduction of human hematopoietic progenitor cells.

Gene therapy using hematopoietic stem cells: Sisyphus approaches the crest.

Gene transfer targeting cells of the blood and immune system was one of the first areas of investigation in the field of gene therapy. Despite the encouraging results achieved in early studies using murine bone marrow, the task of gene transfer into human hematopoietic stem cells proved to be far more difficult. As a result, progress has been disappointingly slow and initial clinical trials generally failed to achieve significant levels of gene marking. The continued application of new advances in vectorology and hematopoietic stem cell biology has now led to improvements in preclinical models that are being translated into clinical trials. The progress and remaining problems are discussed in this review article.

Comparison of the effects of growth factors on retroviral vector-mediated gene transfer and the proliferative status of human hematopoietic progenitor cells.

We have examined the ability of the recombinant hematopoietic growth factors (HGF) interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase retroviral vector-mediated gene transfer into human hematopoietic progenitor cells (HPC). The efficiency of neo gene transfer by the N2 vector into human HPC was enhanced by preculture with either GM-CSF or IL-3 (but not IL-6) and with each combination of the three factors. The combination of IL-3 plus IL-6 consistently produced significantly higher levels of G418-resistant colonies (50-60%) than any of the other combinations of HGF tested. Following preculture with HGF and transduction by N2, marrow was maintained in long-term bone marrow culture (LTBMC) for 2 months. The levels of G418-resistant HPC remained stable, and no apparent depletion of total HPC content resulted from the prior exposure to highly stimulatory doses of factors. The proliferative status of the HPC, following exposure to the HGF, was measured as the percentage of HPC that were inhibited from forming colonies by exposure to the S-phase-specific drug, hydroxyurea. The ability of the different HGF to increase the rate of gene transfer by N2 correlated significantly with the extent to which they stimulated HPC proliferation. These results suggest that the mechanism by which HGF increase rates of gene transfer into HPC is by stimulating cell proliferation. Techniques that produce high rates of gene transfer into long-lived human HPC will facilitate studies to quantitate expression of exogenous genes in hematopoietic cells and may be applicable to clinical gene therapy.

High-resolution analysis of cytosine methylation in the 5long terminal repeat of retroviral vectors.

Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.

Combination of CD80 and granulocyte-macrophage colony-stimulating factor coexpression by a leukemia cell vaccine: preclinical studies in a murine model recapitulating Philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a highly aggressive malignancy caused by the bcr-abl translocation oncogene. To explore alternative treatments for Ph+ ALL we tested gene-modified cell vaccines in the BALB/c-derived BM185 leukemia model. We compared the efficacy of BM185 cell vaccine expressing CD80 alone or in combination with IL-2 or GM-CSF. Mice injected with viable BM185 leukemia cells modified to express CD80 and GM-CSF (BM185/CD80+GM-CSF) showed the highest leukemia rejection rates. Cell vaccines consisting of irradiated BM185/CD80+GM-CSF cells administered subcutaneously stimulated a potent cytotoxic T lymphocyte (CTL) response against parental BM185. Histological examination of the vaccination site showed a large concentration of immune cells. Administration of the BM185/CD80+GM-CSF cell vaccine before intravenous challenge with parental cells caused strong inhibition of leukemia development. Vaccination after subcutaneous challenge with BM185 cells caused efficient elimination of leukemia promoting 40-60% long-term survival rates. The immunization efficacy of the BM185/CD80+ GM-CSF cell vaccine was directly correlated with the percentage of cells expressing the transgenes. In all, this preclinical study shows that leukemia cell vaccines coexpressing CD80 and GM-CSF can potentially be explored for immunotherapy in Ph+ ALL patients.

Expression of biologically active human factor IX in human hematopoietic cells after retroviral vector-mediated gene transduction.

Gene therapy is a potential treatment for hemophilia, wherein cells transduced with a normal factor IX gene could provide a continuous in vivo source of circulating factor IX. In this study, we examined the potential use of hematopoietic cells as a target for factor IX gene therapy. Human myeloid leukemia cells (HL-60) were transduced by retroviral vectors carrying a normal human factor IX cDNA under control of either the Moloney murine leukemia virus long terminal repeat (MoMuLV LTR) (LIXSN), the SV40 promoter (LNSVIX), or a cytomegalovirus (CMV) promoter (LNCIX). Factor IX production was measured in the transduced cells both in the uninduced state and after induction of granulocytic differentiation [with dimethylsulfoxide (DMSO)] or monocytoid differentiation [with phorbol myristic acetate (PMA)]. Transcription of factor IX from the MoMuLV LTR was seen in all cells, with a two-fold increase upon differentiation. Induction with PMA led to an 8- to 15-fold increase in factor IX transcripts from an internal CMV promoter. No factor IX transcripts from the internal SV40 promoter were detected. Immunoreactive factor IX protein was identified by Western blot from induced HL-60 cells transduced by either LIXSN or LNCIX. Factor IX production by HL-60 cells transduced by LNCIX ranged from 38-93 ng/10(6) cells/24 hr following induction of monocytic differentiation. The factor IX antigen titer was directly related to factor IX coagulant titer (r = 0.98; p < 0.001). These data indicate that human myelomonocytic cells are capable of performing the necessary post-translational modifications to produce functional factor IX.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous use of two retroviral vectors in human gene marking trials: feasibility and potential applications.

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors--LNL6 and G1Na--were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD(8+)- and CD(4+)-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10(5) TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.

Suitability of bone marrow from HIV-1-infected donors for retrovirus-mediated gene transfer.

Bone marrow samples from 21 human immunodeficiency virus type 1 (HIV-1)-infected subjects were evaluated for their suitability for retrovirus-mediated gene transduction with anti-HIV-1 genes. The percentages of CD34+ cells that could be isolated from the mononuclear fraction of bone marrow samples were determined. Fifteen of the 21 marrow samples had normal percentages of CD34+ cells isolated by immunomagnetic methods. All seven donors with CD4 counts > 100/mm3 had normal percentages of CD34+ cells; of 14 patients with low CD4 cell counts (< 100/mm3), 5 had reduced and 9 had normal percentages of CD34+ cells. Samples of the marrow were plated in a methylcellulose colony-forming unit (CFU) assay to determine the clonogenic capacity of the progenitor cells. Overall, the marrow samples from HIV-infected donors showed a 44% reduction in CFU derived from the mononuclear cell fraction and a 75% reduction in CFU derived from the isolated CD34+ cell fraction, when compared to marrow samples from uninfected donors. Isolated CD3+ cells were transduced with retroviral vectors containing various anti-HIV-1 genes to determine their susceptibility to gene transfer. Transduction of the clonogenic CD34+ cells by retroviral vectors did not differ among marrow samples from 13 HIV-1+ donors and 9 uninfected donors. Long-term bone marrow cultures established from the transduced CD34+ cells demonstrated equivalent survival of clonogenic progenitor cells from both HIV-1-infected and uninfected marrows. Toxicity from expression of the anti-HIV-1 genes was not observed; the percentages of clonogenic progenitor cells that survived in cultures transduced by vectors carrying anti-HIV-1 genes were similar to those transduced by the control LN vectors. Stromal cells cultured from marrow samples from HIV-1-infected donors showed similar growth kinetics, hematopoietic support function, and enhancement of retrovirus-mediated transduction of CD34+ cells as seen with stromal cells cultured from uninfected marrow donors. Semi-quantitative polymerase chain reaction (PCR) was performed before and after ex vivo transduction to determine the frequency of HIV-1-containing cells in the CD34+ cell preparations. Although HIV-1+ cells were present at low levels in the mononuclear cell fractions of some of the marrow samples, the CD34+ cell preparation from only one marrow sample contained detectable HIV-1 positive cells (< 1 positive cell/100,000 by PCR) prior to transduction. None of the CD34+ cell preparations contained detectable HIV-1 after transduction. These studies demonstrate that HIV-1-infected patients are candidates for retrovirus-mediated transduction of anti-HIV-1 genes in bone marrow gene therapy clinical trials.