Gene-modified Hematopoietic Stem Cells Research Articles

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Integration-Mediated Activation of PRDM16 and HMGA2 in Multiple Clones without Adverse Hematopoietic Consequences Following Transplant of Autologous MGMTP140K Gene-Modified CD34+ Cells

Abstract 2053▪▪This icon denotes a clinically relevant abstractDrug resistance gene therapy with mutant MGMTP140K gene-modified hematopoietic CD34+ cells has been proposed to circumvent myelosuppression associated with alkylating agent chemotherapy; however, the safety of this approach has not yet been demonstrated. We have achieved successful engraftment of MGMTP140K gene-modified hematopoietic progenitor cells in 3 glioblastoma patients following BCNU (600mg/m2) conditioning, as part of a Phase I/II clinical study which includes post-transplant combination O6-benzylguanine (O6BG) and temozolomide (TMZ) chemotherapy. Two of these patients, one of whom remains alive with no evidence for disease progression at more than 2 years since diagnosis, received a total of 9 and 4 cycles of O6BG/TMZ chemotherapy, respectively. We observed transient increases in the number of circulating gene-modified white blood cells (WBCs) and granulocytes in these patients following each cycle. Analysis of CD34+ colony forming cells (CFCs) in peripheral blood revealed increases in the number of gene-modified CFCs over time and with multiple cycles of chemotherapy. Given this, we have conducted a longitudinal retroviral integration site (RIS) analysis in an attempt to identify any selective advantage for gene-modified clones following repeated O6BG/TMZ regimens. In the first patient, at day 200 following transplant, 2 separate RISs were identified, each mapping to the intronic region between exons 1 and 2 of the human PRDM16 gene, previously identified as being associated with insertional activation and clonal expansion in a gene therapy trial for X-linked chronic granulomatous disease (Ott et al 2006). Semi-quantitative capture site frequency analysis revealed that these 2 clones with PRDM16 integrations constituted >20% of the gene-modified cell pool, which included hundreds of RISs at this time point in this patient. Overall gene marking at this same time point was 39.9% of peripheral blood WBCs, indicating that as many as 8% of circulating WBCs arose from these two clones. In the second patient, 3 distinct RISs were identified mapping to the 3’untranslated region (UTR) of the HMGA2 gene, within a cluster of let-7 microRNA binding domains known to be responsible for post-transcriptional regulation of this gene product. While HMGA2-associated RISs have been reported in 2 other gene therapy trials for β-thalassemia (Cavazzana-Calvo et al 2010) and X-linked severe combined immunodeficiency syndrome (Wang et al 2010), the genomic loci of these RISs mapped to the third intron of this genomic sequence. Capture frequency analysis indicated that overall contribution of the most abundant HMGA2-associated clone identified in this study reached 5.2% of gene-modified peripheral blood WBCs at day 300 after transplant. Analysis of PRDM16 and HMGA2 transcripts in each patient’s cells by reverse-transcriptase real-time PCR indicated increased transcription of these genes in peripheral blood and bone marrow WBCs. Analysis of WBC subsets in the first patient found PRDM16 RISs present in granulocyte and CD3+ blood cell lineages, suggesting these clones originated from multipotential gene-modified hematopoietic stem cells capable of differentiating despite PRDM16 transactivation. Furthermore, since discontinuation of chemotherapy, we have observed a steady decline in levels of both PRDM16 clones, with complete disappearance of one clone. Extended analysis of HMGA2 expression and clone tracking in WBC subsets of the second patient is currently under way. Importantly, analysis of both patients bone marrow demonstrated normal cytogenetics, with no evidence for MDS or leukemia by hematopathology and flow cytometry. We believe that these data, in combination with other clinical gene therapy studies identifying similar clones, indicate the PRDM16 and HMGA2 gene sequences as “hot-spots” for either initial retrovirus integration in CD34+ hematopoietic progenitor and stem cells, or as genomic loci where aberrant gene expression, as a result of retrovirus integration, is associated with increased cell proliferation. At this time, these observations suggest that in the absence of underlying genomic disease, in vivo fluctuations in PRDM16- and HMGA2-associated clones following transplant may be a common, clinically inconsequential phenomenon in retrovirus-mediated gene therapy targeting CD34+ cell populations for modification. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic stem cell expansion and gene therapy

Hematopoietic stem cell (HSC) gene therapy remains a highly attractive treatment option for many disorders, including hematologic conditions, immunodeficiencies including human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), and other genetic disorders such as lysosomal storage diseases. In this review, we discuss the successes, side-effects and limitations of current gene therapy protocols. In addition, we describe the opportunities presented by implementing ex vivo expansion of gene-modified HSC, as well as summarize the most promising ex vivo expansion techniques currently available. We conclude by discussing how some of the current limitations of HSC gene therapy could be overcome by combining novel HSC expansion strategies with gene therapy.

Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice

AbstractInherited hematologic defects that lack an in vivo selective advantage following gene correction may benefit from effective yet minimally toxic cytoreduction of endogenous hematopoietic stem cells (HSCs) prior to transplantation of gene-modified HSCs. We studied the efficacy of administering a novel sequential treatment of parenteral ACK2, an antibody that blocks KIT, followed by low-dose irradiation (LD-IR) for conditioning of wild-type and X-linked chronic granulomatous disease (X-CGD) mice. In wild-type mice, combining ACK2 and LD-IR profoundly decreased endogenous competitive long-term HSC repopulating activity, and permitted efficient and durable donor-derived HSC engraftment after congenic transplantation. ACK2 alone was ineffective. The combination of ACK2 and LD-IR was also effective conditioning in X-CGD mice for engraftment of X-CGD donor HSCs transduced ex vivo with a lentiviral vector. We conclude that combining ACK2 with LD-IR is a promising approach to effectively deplete endogenous HSCs and facilitate engraftment of transplanted donor HSCs.

Update on gene therapy for adenosine deaminase-deficient severe combined immunodeficiency

The present review describes the recent progress in the treatment of adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) using autologous gene-modified hematopoietic stem cells, comparing immune reconstitution with respect to allogeneic transplant and discussing differences with gene therapy for SCID-X1. Since 2000, more than 30 ADA-SCID patients have been treated with gene therapy worldwide, with successful outcome in most cases, consisting of progressive immune reconstitution, efficient systemic detoxification, and long-term multilineage engraftment. Gene therapy resulted in restoration of thymic activity and T-cell functions, although the kinetic of reconstitution was slower compared with standard bone marrow transplant. Unlike allogeneic transplant from alternative donors, survival after gene therapy is excellent. In comparison with SCID-X1, ADA-SCID gene therapy presents a better safety profile and engraftment of multilineage transduced stem/progenitor cells, thanks to the use of nonmyeloablative preconditioning. New approaches using safer integrating vectors are being developed, which may lead to safer and effective gene therapy for ADA-SCID and other genetic disorders. In the last decade, gene therapy has been developed as a successful and safe alternative strategy for patients affected by ADA-SCID lacking a compatible sibling donor. The application of innovative vector technology might further improve its efficacy and safety profile.

522. Non-Myeloablative Conditioning Regimen To Increase Engraftment of Gene-Modified Hematopoietic Stem Cells

522. Non-Myeloablative Conditioning Regimen To Increase Engraftment of Gene-Modified Hematopoietic Stem Cells

Development of gene therapy: potential in severe combined immunodeficiency due to adenosine deaminase deficiency

The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID) and especially adenosine deaminase (ADA)-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a protocol for the autologous transplant of retroviral vector-mediated gene-modified hematopoietic stem cells, which has proved to be both successful and, to date, safe. Patients in trials in three different countries have shown long-term immunological and metabolic correction. Nevertheless, improvements to the safety profile of viral vectors are underway and will undoubtedly reinforce the position of stem cell gene therapy as a treatment option for ADA-SCID.

Efficient MGMTP140K-Mediated In Vivo Selection and Chemoprotection of Long-Term Repopulating Cells in Nonhuman Primates Following Reduced Intensity Conditioning.

Abstract 3572Poster Board III-509Strategies using gene-modified hematopoietic stem cells to treat various severe hematopoietic diseases, including but not limited to hemoglobinopathies, will likely require high levels of gene marking. Here we have established efficient and stable in vivo selection in nonhuman primates using methylguanine methyltransferase (MGMTP140K). In the macaque (Macaca nemestrina) we were able to increase pre-chemotherapy lentiviral gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. Furthermore, stable increases in gene marking were also observed in red blood cells (RBCs) and platelets (PLTs) with a pre-chemotherapy gene marking level of 5.6% and 6.7%, respectively, and a post-chemotherapy gene marking level of 15.2% and 64.0%, respectively. Importantly, the chemotherapy regimen was well tolerated, and engraftment was polyclonal as determined by analyzing long-term repopulating clones by LAM-PCR. In order to minimize extra-hematopoietic toxicity we have began to test a more clinically applicable conditioning regimen in the macaque model. This reduced intensity conditioning regimen should allow treatment of patients with severe hematopoietic or infectious diseases, who may not tolerate a high dose conditioning regimen. We tested targeted busulfan for conditioning to provide sufficient myelosuppression and to facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene modified cells (∼1.7 × 107 CD34-selected cells/kg), there was moderate cytopenia with ANC <500/mL for 7 days and thrombocytopenia with a nadir of 18,000/mL. Following stable hematopoietic recovery, we observed gene marking, determined by RT-PCR, in total white blood cells as a provirus copy number of 0.04 (∼4% gene marking) that, following a single cycle of O6BG (x2) and BCNU, rose to 0.16 (∼16% gene marking). Currently, gene marking has been stable for more than 9 months following chemotherapy. The treatment was well tolerated with only transient elevated liver enzymes following O6BG/BCNU treatment and no additional extra-hematopoietic toxicity has been observed. Clonality studies before and after in vivo selection is underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach. In summary, we have attained efficient and stable in vivo selection of long-term repopulating cells in nonhuman primates, and have extended this approach to use a reduced intensity conditioning regimen that should be well tolerated in patients with many hematopoietic diseases. Disclosures:No relevant conflicts of interest to declare.

A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction

AbstractWe show that lentiviral delivery of human γ-globin gene under β-globin regulatory control elements in hematopoietic stem cells (HSCs) results in sufficient postnatal fetal hemoglobin (HbF) expression to correct sickle cell anemia (SCA) in the Berkeley “humanized” sickle mouse. Upon de-escalating the amount of transduced HSCs in transplant recipients, using reduced-intensity conditioning and varying gene transfer efficiency and vector copy number, we assessed critical parameters needed for correction. A systematic quantification of functional and hematologic red blood cell (RBC) indices, organ pathology, and life span was used to determine the minimal amount of HbF, F cells, HbF/F-cell, and gene-modified HSCs required for correcting the sickle phenotype. We show that long-term amelioration of disease occurred (1) when HbF exceeded 10%, F cells constituted two-thirds of the circulating RBCs, and HbF/F cell was one-third of the total hemoglobin in sickle RBCs; and (2) when approximately 20% gene-modified HSCs repopulated the marrow. Moreover, we show a novel model using reduced-intensity conditioning to determine genetically corrected HSC threshold that corrects a hematopoietic disease. These studies provide a strong preclinical model for what it would take to genetically correct SCA and are a foundation for the use of this vector in a human clinical trial.

Cotransplantation with MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation in nonhuman primates

Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.

Cell-Intrinsic Heterogeneity Plays An Important Role in the Development of Clonal Dominance after Retroviral Gene Transfer into Hematopoietic Stem Cells

The development of clonal imbalance after transplantation of genetically modified hematopoietic cells is a cause of concern in the long-term follow-up of patients undergoing gene therapy for the treatment of severe or acquired hematopoietic disorders. We and others have previously described how insertional proto-oncogene dysregulation by transgene integration may provoke clonal restriction and leukemia, thus becoming a dose-limiting toxicity of gene therapy. When targeting populations enriched for or depleted from hematopoietic stem cells (HSC) in the C57Bl6 CD45 chimerism model, we found that intrinsic stem cell potential is a conditio sine qua non for the establishment of expanding insertional mutants. Mice observed for 6–7 months after co-transplantation of gene-modified cells and non-transduced fresh competitor cells were monitored in regular intervals of 6 weeks and the emergence of dominant clones was assessed by flow cytometry in combination with an LM-PCR procedure validated on mixtures of polyclonal and oligoclonal DNA. Dominant clones originating after gammaretroviral insertion in the Evi1 locus reproducibly occurred with a frequency of 1:10,000 when targeting multipotent LSK cells or short-term repopulating HSC (LSK CD34+ CD135−), but no such events were detected in the progeny of >1 million Sca1- Lin- c-Kit+ (LK) cells or ~75,000 multipotent progenitor cells (MPP, LSK CD34+ CD135+). Dominant clones originating from multipotent cells and displaying insertional upregulation of Evi1 showed greatly diminished T lymphopoiesis in vivo, formally demonstrating transforming events. Residual progeny of MPP or LK cells was detected in transplanted animals with insertional events in proto-oncogenes, but these clones were unable to expand to significant levels of hematopoiesis (>1%). Targeting HSC-enriched cell populations (LSK CD34+ CD135− or LSK CD34− CD135−), a comparison of gamma-retroviral transduction conditions in a 5 days serum-free culture period and lentiviral transduction in a 20h protocol revealed that the latter conditions significantly improved chimerism with a greatly increased clonal diversity in the first 8 weeks of repopulation. However, after lentiviral transduction clonal dominance progressively developed over an observation time of 6 months, although there was no evidence for insertional proto-oncogene upregulation as the underlying cause even when using a lentiviral vector with a strong internal enhancer-promoter capable of insertional long-distance effects. Our study suggests two important conclusions: (1) Insertional mutagenesis in gene therapy is unlikely to endow differentiating progenitor cells with (leukemogenic) stem cell potential and (2) clonal restriction developing in the long-term follow-up after transplantation of gene-modified hematopoietic stem cells is not necessarily a side effect of insertional mutagenesis, but may also reflect classical “gene marking” of a stem cell clone with a strong intrinsic potential for competitive dominance.

Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

Retroviral-mediated delivery of the P140K mutant O(6)-methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6)-benzylguanine) of endogenous MGMT. Because detoxification of O(6)-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMT(P140K) activity, we show that MGMT(P140K) expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMT(P140K) activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-alkylguanine adducts. Moreover, very high expression of MGMT(P140K) was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT(P140K), but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT(P140K) to the nucleus/chromatin. These data show that very high expression of MGMT(P140K) has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT(P140K), whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.

Hematopoietic stem cell–targeted neonatal gene therapy reverses lethally progressive osteopetrosis in oc/oc mice

AbstractInfantile malignant osteopetrosis (IMO) is a fatal disease caused by lack of functional osteoclasts, and the only available treatment is hematopoietic stem cell (HSC) transplantation. In the majority of patients, the TCIRG1 gene, coding for a subunit of a proton pump essential for bone resorption, is mutated. Oc/oc mice have a deletion in the homologue gene (tcirg1) and die at 3 to 4 weeks, but can be rescued by neonatal transplantation of HSCs. Here, HSC-targeted gene therapy of osteopetrosis in the oc/oc mouse model was developed. Oc/oc fetal liver cells depleted of Ter119-expressing erythroid cells were transduced with a retroviral vector expressing tcirg1 and GFP, and subsequently transplanted intraperitoneally to irradiated neonatal oc/oc mice. Eight of 15 mice survived past the normal life span of oc/oc mice. In vitro osteoclastogenesis revealed formation of GFP-positive osteoclasts and bone resorption, albeit at a lower level than from wild-type cells. The skeletal phenotype was analyzed by X-ray and histopathology and showed partial correction at 8 weeks and almost normalization after 18 weeks. In summary, osteopetrosis in oc/oc mice can be reversed by neonatal transplantation of gene-modified HSCs leading to long-term survival. This represents a significant step toward the development of gene therapy for osteopetrosis.

In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer

SCID patients have been successfully treated by administration of ex vivo gene-corrected stem cells. However, despite its proven efficacy, such treatment carries specific risks and difficulties. We hypothesized that some of these drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. Indeed, in vivo intrathymic transfer of a gene that provides a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny, allowing long-term immune reconstitution. We assessed the feasibility of this approach in a murine model of ZAP-70-deficient SCID. A T cell-specific ZAP-70-expressing lentiviral vector was injected into thymi of adult ZAP-70-/- mice without prior conditioning. This resulted in the long-term differentiation of mature TCR-alphabeta+ thymocytes, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells demonstrated a partially diversified receptor repertoire and were responsive to alloantigens in vitro and in vivo. Improved treatment efficacy was achieved in infant ZAP-70-/- mice, in which the thymus is proportionately larger and a higher percentage of prothymocytes are in cycle. Thus, intrathymic injection of a lentiviral vector could represent a simplified and potentially safer alternative to ex vivo gene-modified hematopoietic stem cell transplantation for gene therapy of T cell immunodeficiencies.

Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4

To attain therapeutic levels of gene-modified hematopoietic stem cells, it may be necessary in the majority of disorders to provide an in vivo selective advantage that facilitates the expansion of their numbers. A popular strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O6-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified hematopoietic stem cells through the delivery of the homeobox transcription factor HOXB4. By developing a novel tricistronic retroviral vector, we have facilitated the simultaneous coexpression of a mutant version of MGMT and HOXB4 in retrovirally transduced bone marrow. Using an in vivo competitive repopulation assay, we demonstrate that primary bone marrow cells containing this construct show enhanced reconstitution following transplant and improved selection subsequent to chemotherapeutic challenge in comparison to cells expressing either HOXB4 or MGMT alone. This selection advantage was evident even when HOXB4/MGMT-coexpressing cells were infused along with a large excess of unmodified cells. We propose that this selection cassette may facilitate the in vivo expansion of gene-modified hematopoietic stem cells at a level in excess of previous strategies.

Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction.

Autologous transplantation of gene-modified hematopoietic stem cells may provide a therapeutic strategy for several monogeneic disorders. In previous studies, retroviral gene transfer of coagulation factor VIII (FVIII) into FVIII(-/-) mouse bone marrow (BM) cells did not result in detectable plasma FVIII levels. However, specific immune tolerance was achieved against neo-antigenic FVIII. Here, we used lentiviral vectors to study the ability of various hematopoietic cell types to synthesize and secrete recombinant FVIII. Several myeloid, monocytic and megakaryocytic cell lines (K-562, TF-1, Monomac-1, Mutz-3, Meg-01) expressed FVIII at 2-12 mU/10(4) cells. In contrast, two lymphatic cell lines, BV-173 and Molt-4, were less-efficiently transduced and did not express detectable FVIII. Similarly, peripheral blood-derived primary monocytes were transduced efficiently and expressed up to 20 mU/10(4) cells, whereas primary lymphocytes did not express FVIII. Although human and canine CD34(+) cells were transduced efficiently, the cells expressed very low levels of FVIII (up to 0.8 mU/10(4) cells). Following xenotransplantation of transduced CD34(+) into NOD/SCID mice, ELISA failed to detect FVIII in the plasma of engrafted mice. However, NOD/SCID repopulating cell (SRC)-derived human monocytes isolated from BM of these mice secreted functional recombinant FVIII after culture ex vivo. Again, SRC-derived human lymphocytes did not secrete FVIII. Therefore, certain hematopoietic cell types are able to synthesize and secrete functional recombinant FVIII. Our results show for the first time that transplantation of transduced CD34(+) progenitors may give rise to differentiated hematopoietic cells secreting a nonhematopoietic recombinant protein.

Gene therapy-based treatment for HIV-positive patients with malignancies.

Gene therapy for the treatment of HIV has long been a goal of many investigators. The majority of trials have involved the use of lymphocytes transduced with vectors promoting resistance to HIV infection or replication. Unfortunately, the results have been less than encouraging with low-level marking and, more importantly, clearance of these lymphocytes from the circulation. Conversely, gene-modified hematopoietic stem cells appear able to introduce foreign transgenes while avoiding immunologic clearance. Furthermore, the use of less toxic conditioning regimens for allogeneic transplantation provides an attractive approach to conferring HIV resistance while allowing treatment of HIV-related disorders such as malignancies. This combination of nonmyeloablative allogeneic transplantation using gene-modified hematopoietic stem cell theoretically overcomes the high transplant mortality associated with traditional conditioning regimens in patients with HIV as well as providing a self-renewing source of HIV-resistant cells. To assess the safety and feasibility of such an approach, a clinical protocol was initiated in those patients infected with HIV with a hematologic malignancy meeting the standard indications for allogeneic transplantation and provided here is an update to the previously published original report. Only patient 1 received genetically modified cells. Both patients tolerated the procedure with no effect on viral load and improved CD4 counts, and patient 1 remains in complete remission from acute myelogenous leukemia 3 years post transplant. Patient 2 also achieved clinical remission from chemorefractory Hodgkin's disease but died of relapsed disease 12 months after transplantation. Vector-transduced cells remain detectable at low levels more than 3 years post-transplantation, suggesting the potential for gene therapy as a reasonable goal for the treatment of HIV.

Adenosine deaminase gene therapy protocol revisited.

Adenosine deaminase gene therapy protocol revisited.

An improved vector for high-level, consistent retroviral transgene expression in human thymocytes after competitive reconstitution from transduced peripheral blood stem cells.

One problem in hematopoietic stem cell (HSC)-based gene therapy is the low-level, and often transient, transgene expression in progeny cells in vivo. Here we have evaluated retroviral vector designs for improved long-term in vivo transgene expression levels in thymocytes recovered after transplantation of gene-modified HSCs. First, several vector designs were screened in vitro by single-cell analysis of transgene marking and expression to rapidly identify optimal vectors for sensitive tracking of marked cells. Next, using one optimal vector, we show that gene-modified HSCs can competitively reconstitute thymopoiesis in SCID-hu thymus/liver mice, with transgene expression detectable on 0-40% of marked donor thymocytes. Modified vector designs (termed MSCV-SAR and MoMLV-SAR), which enhance transgene expression in primary T cells in vitro, were shown here to improve in vivo transgene expression levels per cell 12- to 14-fold (mean fluorescence intensity was 2175 for MSCV-SAR vs. 174 for LNGFRSN; %NGFR(+) donor(+) cells with high-level expression was 58% for MSCV-SAR vs. 4% for LNGFRSN). Importantly, 61% of grafts had high-level transgene expression on thymocytes with the MSCV-SAR vector versus 0% of grafts for LNGFRSN or MoMLV-SAR. Transgene expression was demonstrated in various stages of thymocyte differentiation and was consistently detected in early thymic progenitors. We suggest that the MSCV-SAR vector described here is particularly advantageous for applications requiring high-level, consistent transgene expression in a diverse repertoire of T cells derived from gene-modified HSC grafts.

Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy.

Retroviral vector-mediated expression of the homeoboxgene, HoxB4, in hematopoietic cells has been reported to mediate a benign expansion of gene-modified hematopoietic stem and precursor cells in vivo. In the present study, we used a novel coexpression strategy for coordinated expression of HoxB4 along with a cytoplasmic protein from a retroviral vector. The novel coexpression strategy, based on cotranslational protein separation mediated by the 2A sequence of foot-and-mouth disease virus (FMDV), allows an indirect quantification of HoxB4 expression levels when inserting a reporter such as the enhanced green fluorescent protein (GFP) in the retroviral vector. Presence of the 2A sequence did not interfere with the correct subcellular localization of HoxB4 (nuclear) and GFP (cytoplasmic), nor with the titer of bicistronic vectors, and mediated functional long-term coexpression (at least 1 year) of GFP and HoxB4 after transplantation of transduced mouse bone marrow cells in mice.