Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-{alpha} on Fanconi anemia hematopoietic stem and progenitor cells.

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha, we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.

Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI.

BACKGROUND: A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O6-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. RESULTS: All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was approximately 4 times greater than that of an IRES based vector. CONCLUSION: The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

Lentivirus-mediated expression of mutant MGMTP140K protects human CD34+ cells against the combined toxicity of O6-benzylguanine and 1,3-bis(2-chloroethyl)-nitrosourea or temozolomide.

Lentiviral vectors are capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. We constructed human immunodeficiency virus type 1-based self-inactivating lentiviral vectors to express either wild-type or an O6-benzylguanine (O6-beG)-resistant mutant form of the human O6-alkylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:[protein]-L-cysteine S-methyltransferase, EC 2.1.1.63) and transduced K562 and granulocyte colony-stimulating factor-mobilized human peripheral blood CD34+ cells. After transduction, K562 cells expressed high levels of MGMT as determined by Western blot, immunocytochemistry, and biochemical assay. A colony-forming survival assay showed significant protection against O6-beG plus 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) or temozolomide (TMZ) toxicity. Similarly, a single transduction of CD34+ cells resulted in a 13- to 14-fold increase in the level of MGMT expression. In comparison with non-transduced cells, mutant MGMTP140K-transduced CD34+ cells showed significant resistance against the combined toxicity of O6-beG with either TMZ or BCNU: there was an approximately 9-fold increase in the survival of colony-forming cells as indicated by the IC50 values after O6-beG plus TMZ treatment and an approximately 5-fold increase in the case of O6-beG plus BCNU treatment. These results show that lentivirus-mediated expression of MGMTP140K can efficiently protect the hematopoietic compartment against the combined toxicity of O6-beG plus TMZ or BCNU.

Genetic manipulation of drug sensitivity in haematopoietic cells.

The haematopoietic system can be manipulated genetically to increase either its resistance to drugs or its sensitivity to certain agents. Gene transfer and expression of specific drug-resistance factors might protect haematopoietic function during antitumour chemotherapy, or allow enrichment of gene-modified cells in vivo. By contrast, gene transfer of a prodrug activator, to confer sensitivity to otherwise nontoxic prodrugs, might allow deletion of engrafted cells in the event of an adverse effect such as graft-versus-host disease or the induction of a neoplasm. In addition, expression of a prodrug activator in tumour-infiltrating haematopoietic cells could provide a means of specifically activating a cytotoxic agent within a tumour mass.

Total deletion of in vivo telomere elongation capacity: an ambitious but possibly ultimate cure for all age-related human cancers.

Despite enormous effort, progress in reducing mortality from cancer remains modest. Can a true cancer "cure" ever be developed, given the vast versatility that tumors derive from their genomic instability? Here we consider the efficacy, feasibility, and safety of a therapy that, unlike any available or in development, could never be escaped by spontaneous changes of gene expression: the total elimination from the body of all genetic potential for telomere elongation, combined with stem cell therapies administered about once a decade to maintain proliferative tissues despite this handicap. We term this therapy WILT, for whole-body interdiction of lengthening of telomeres. We first argue that a whole-body gene-deletion approach, however bizarre it initially seems, is truly the only way to overcome the hypermutation that makes tumors so insidious. We then identify the key obstacles to developing such a therapy and conclude that, while some will probably be insurmountable for at least a decade, none is a clear-cut showstopper. Hence, given the absence of alternatives with comparable anticancer promise, we advocate working toward such a therapy.

The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine.

The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.

Dual agent chemoprotection by retroviral co-expression of either MDR1 or MRP1 with the P140K mutant of O6-methylguanine-DNA-methyl transferase.

Tumour resistance to chemotherapeutic agents results in most chemotherapy being administered in a multi-agent fashion that is often associated with a high level of toxicity in highly proliferative tissues such as the haematopoietic compartment. Thus, whilst many genetic manipulation strategies aim to protect normal tissue against a single component of a multi-agent regime, it is clearly preferable to protect normal cells against all toxicities. In this study we have used retroviral gene transfer to achieve co-expression of either p-glycoprotein (MDR1) or multi-drug resistance-related protein 1 (MRP1) with the P140K mutant form of O6-methylguanine-DNA-methyl transferase (MGMT) which, unlike the wild-type protein, is insensitive to inactivation by tumour sensitisers such as O6-benzylguanine (O6-BeG) or PaTrin2. The combination of certain MDR1/MRP1 substrate drugs with O6-alkylating agents (against which MGMT confers resistance) is particularly myelotoxic. We show here that haematopoietic progenitors co-expressing mutant MGMT with an ABC-transporter exhibit resistance to combination chemotherapy in vitro. This combination of drug transporter and DNA repair function may provide an effective in vivo protection of the haematopoietic compartment during tumour ablation using combination chemotherapy.

Radioprotective gene therapy through retroviral expression of manganese superoxide dismutase.

BACKGROUND: Radiotherapy for the control of cancer, either alone or in conjunction with chemotherapy, is often limited by normal tissue toxicity including haematopoietic toxicity. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation-induced cytotoxicity. The antioxidant enzyme manganese superoxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide. METHODS: We have investigated the potential of SOD2 overexpression, through retroviral gene transfer using a retrovirus optimized for transcription in early haematopoietic cells, to enhance the radioresistance of a human erythroleukaemic cell line and primary murine bone marrow. Using these as in vitro models we have investigated whether SOD2 gene therapy may be suitable for the protection of the haematopoietic compartment from the effects of ionizing radiation. RESULTS: Here we demonstrate using both biological and physical assays that overexpression of SOD2 protects haematopoietic cells from ionizing radiation injury. Our results show that an increase in the levels of SOD2 enzymatic activity within K562 cells (from 160.7 +/- 23.6 to 321.8 +/- 45.2 U/mg protein) or primary murine haematopoietic progenitor cells leads to both a significant decrease in DNA fragmentation and a significant increase in clonogenic survival, as evident by a significant increase in Dbar (from 2.66 to 3.42Gy), SF2 (from 0.52 to 0.73) values, and a significant decrease in the alpha value (from 0.3040 +/- 0.037 to 0.0630 +/- 0.037 Gy(-1)) when compared either to cells transduced with a retroviral vector encoding eGFP alone or to the parental line. CONCLUSIONS: The results presented suggest that retroviral radioprotective gene therapy may be applicable to the haematopoietic compartment, enabling radiation dose escalation in cancer therapy.

Protection and selection for gene therapy in the hematopoietic system.

Hematopoietic stem cell gene therapy is potentially curative for a number of inherited and acquired disorders. However, poor gene transfer and expression in repopulating hematopoietic stem cells attenuate this potential. Here we review potential means of conferring a selective advantage to hematopoietic stem cells and their progeny, and discuss the issues that surround the use of selective advantages in vivo.

High transduction efficiency of circulating first trimester fetal mesenchymal stem cells: potential targets for in utero ex vivo gene therapy.

We recently reported the existence of fetal mesenchymal stem cells in first trimester fetal blood. Here we demonstrate that fetal mesenchymal stem cells from as early as eight weeks of gestation can be retrovirally transduced with 99% efficiency without selection. Circulating fetal mesenchymal stem cells are known to readily expand and differentiate into multiple tissue types both in vitro and in vivo, and might be suitable vehicles for prenatal gene delivery. With advances in early fetal blood sampling techniques, we suggest that genetic disorders causing irreversible damage before birth could be treated in utero in the late first/early second trimester by genetically manipulated autologous fetal stem cells.

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.

Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion.

Human marrow stromal cells (MSCs) can be isolated from bone marrow and differentiate into multiple tissues in vitro and in vivo. These properties make them promising tools in cell and gene therapy. The lack of a specific MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion prior to clinical use. This may severely reduce MSC proliferative capacity to the point that the residual proliferative potential is insufficient to maintain long-term tissue regeneration upon reinfusion. In this study we determined the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. We report that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan.

Role of MDR1 and MRP1 in trophoblast cells, elucidated using retroviral gene transfer.

Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [3H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus.

A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow.

Homing of bone marrow stromal cells (MSCs) to bone and bone marrow after transplantation, important for the correction of conditions such as metabolic storage disorders, can occur but with poor efficiency. Substantial improvements in engraftment will be required in order to derive a clinical benefit from MSC transplantation. Chemokines are the most important factors controlling cellular migration. Stromal-derived factor-1 (SDF-1) has been shown to be critical in promoting the migration of cells to the bone marrow, via its specific receptor CXCR4. The aim of our study was to investigate CXCR4 expression on MSCs and its role in mediating migration to bone marrow. We show that CXCR4, although present at the surface of a small subset of MSCs, is important for mediating specific migration of these cells to bone marrow.

Retrovirally mediated correction of bone marrow-derived mesenchymal stem cells from patients with mucopolysaccharidosis type I.

We have investigated the utility of bone marrow-derived mesenchymal stem cells (MSCs) as targets for gene therapy of the autosomal recessive disorder mucopolysaccharidosis type IH (MPS-IH, Hurler syndrome). Cultures of MSCs were initially exposed to a green fluorescent protein-expressing retrovirus. Green fluorescent protein-positive cells maintained their proliferative and differentiation capacity. Next we used a vector encoding alpha-L-iduronidase (IDUA), the enzyme that is defective in MPS-IH. Following transduction, MPS-IH MSCs expressed high levels of IDUA and secreted supernormal levels of this enzyme into the extracellular medium. Exogenous IDUA expression led to a normalization of glycosaminoglycan storage in MPS-IH cells, as evidenced by a dramatic decrease in the amount of (35)SO(4) sequestered within the heparan sulfate and dermatan sulfate compartments of these cells. Finally, gene-modified MSCs were able to cross-correct the enzyme defect in untransduced MPS-IH fibroblasts via protein transfer.