Leslie J. Fairbairn

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.

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

BackgroundA 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.ResultsAll 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 ~4 times greater than that of an IRES based vector.ConclusionThe 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.

Erythroid development of the FDCP-Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3

Summary .Conditions are described which promote the erythroid development of the FDCP‐Mix A4 (A4) cell line with accompanying proliferation of the cells. The requirements for this development are low concentrations of interleukin 3 (IL‐3) plus the presence of erythropoietin (epo) and haemin. When high concentrations of IL‐3 are added with erythropoietin and haemin the cells do not differentiate and maintain their blast cell morphology. Addition of haemin, in the absence of erythropoietin, does not promote erythroid development, but the presence of haemin with erythropoietin promotes increased proliferation and maturation. The morphological maturation of A4 cells along the erythroid lineage is accompanied by a gradual loss of clonogenic potential, loss of A4 cell multipotency, increased erythropoietin receptor expression, and an increased expression of the β‐globin gene. An initial increase in mitogenic responsiveness to erythropoietin is followed by a decrease as the cells become refractory to all mitogenic stimuli with the acquisition of a postmitotic, mature erythroid cell phenotype.

Chemoprotective gene transfer II: multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase.

Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte–macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocytochemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with o6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). this study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an o6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.

Genotoxicity studies on the azo dye Direct Red 2 using the in vivo mouse bone marrow micronucleus test

The clastogenicity of the azo dye Direct Red 2 (DR2) has been investigated using the murine bone marrow micronucleus assay. A potent dose-dependent response was observed following oral gavage of DR2 up to 4 mg/kg, after which significant toxicity to the erythroid compartment was observed. The route of administration had a significant effect on the frequency of micronucleus formation: intraperitoneal injection was approximately two-fold less clastogenic than the equivalent dose delivered orally (p<0.05). The requirement for activation of DR2 by intestinal microflora was indicated by the fact that mice given acid-treated water prior to administration of DR2 showed a significant reduction (40%; p<0.001) in micronucleated polychromatic erythrocyte formation. The implications of these findings for the health and safety of occupationally exposed workers are discussed.

Chemoprotective gene transfer I : Transduction of human haemopoietic progenitors with O6-benzylguanine-resistant O6-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O6-alkylating agents in vitro

Retroviral transduction was used to introduce cDNAs encoding two mutants of human O6-alkylguanine-DNA alkyltransferase (hAT), one of which (hATPA) is 16 times more resistant to O6-benzylguanine (O6-beG), and the other (hATPA/GA) which is almost totally refractory to inactivation relative to the wild-type protein, into K562 human erythroleukaemic cells. A colony-forming assay was used to demonstrate significant protection (P < 0.001) against mitozolomide or temozolomide toxicity in k562 clones expressing either hat mutant, as determined from an in vitro assay of activity. however, protection against these agents was reduced in hatpa expressing cells in the presence of 1 μm o6-beG and was lost in the presence of 20 μM O6-beG while cells expressing hATPA/GA retained protection even in the presence of 20 μM O6-beG (P < 0.001). using primary human cord blood-derived cd34+ haemopoietic cells in which PCR analy-sis indicated that up to 70% of progenitors were trans- duced with retroviral constructs harbouring hATPA/GA, we observed significant protection of the granulocyte–macrophage colony-forming cells against mitozolomide (P < 0.05) and temozolomide (p < 0.001) induced toxicity in the presence of o6-beG. These findings indicate that retrovirus-mediated expression of hATPA/GA in primitive primary human haemopoietic cells is possible and does provide O6-beG-resistant protection for these cells. Using this strategy in patients may simultaneously permit attenuated myelosuppression and increased sensitivity of tumour cells to the effects of O6-alkylating agent chemotherapy. These data, taken together with the study reported by Chinnasamy et al in the accompanying article in this issue showing reduced toxicity and clastogenicity in murine haemopoietic progenitors, make a compelling case to test this strategy clinically.

A novel dual function retrovirus expressing multidrug resistance 1 and O6-alkylguanine-DNA-alkyltransferase for engineering resistance of haemopoietic progenitor cells to multiple chemotherapeutic agents.

Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future.

Expression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.

In order to investigate the importance of 3‐methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3‐methyladenine DNA glycosylase‐1 which specifically removes 3‐alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus‐packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3‐methyladenine DNA glycosylase activity in cell extracts showed an up to 10‐fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N‐methyl‐N‐nitrosourea than their parent cell lines, indicating that 3‐methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3‐methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.

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 O6‐methylguanine‐DNA‐methyltransferase (MGMT) inactivator O6‐benzylguanine (O6‐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 O6‐beG‐insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O6‐(4‐bromothenyl)guanine, PaTrin‐2), which shows potential advantages over O6‐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 P140K 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 MGMTP140K is highly resistant to the cytotoxic effects of PaTrin‐2 in combination with the methylating agent temozolomide, and that cells expressing MGMTP140K can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMTP140K 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 O6‐beG/temozolomide. Thus our data show that MGMTP140K is a suitable candidate for chemoprotective gene therapy where PaTrin‐2 is being used in conjunction with temozolomide. Copyright

New perspectives for cancer chemotherapy by genetic protection of haematopoietic cells.

The effectiveness of anti-cancer chemotherapy can be limited by acute suppression of the bone marrow (myelosuppression). There is also a risk of therapy-related secondary haematopoietic malignancy as well as acute and longer term effects in other tissues. Clinical strategies have been established to address some of these problems, particularly toxic effects on the bone marrow (acute myelotoxicity); however, there is still substantial scope for improving the management of chronic toxicity and mutagenicity to haematopoietic cells and collateral damage to non-haematopoietic cells during chemotherapy. In this review, we have discussed a novel strategy that involves the transfer and expression of drug-resistance functions into haematopoietic stem cells and more-mature blood progenitor cells, to overcome both the acute and long-term deleterious effects of anti-tumour treatment in bone marrow. The potential advantages of this approach include: (1) the in vivo selection of protected cell populations, which offers the possibility of intensification or escalation of chemotherapeutic drug doses; (2) a reduction in the frequency of therapy-related leukaemia and (3) tumour sensitisation to chemotherapy at the same time as haematopoietic protection.