Insertional mutagenesis in gene therapy and stem cell biology

PURPOSE OF REVIEW: Recent preclinical and clinical studies revealed that the semirandom insertion of transgenes into chromosomal DNA of hematopoietic cells may induce clonal competition, which potentially may even trigger leukemia or sarcoma. Insertional mutagenesis caused by gene vectors has thus led to major uncertainty among those developing advanced hematopoietic cell therapies. This review summarizes novel studies of underlying mechanisms; these studies have demonstrated the possibility of improved gene vector biosafety and generated new insights into stem cell biology. RECENT FINDINGS: The characteristic insertion pattern of various retroviral gene vector systems may be explained by properties of the viral integrase and associated cellular cofactors. Cell culture assays and animal models, including disease-specific and cancer-prone mouse models, are emerging that reveal the contributions of vector features and systemic factors to induction of clonal imbalance. Databases summarizing vector insertion sites in dominant hematopoietic clones are evolving as new tools to identify genes that regulate clonal homeostasis. SUMMARY: Mechanistic studies of insertional mutagenesis by random gene vector insertion will lead to improved tools for advanced hematopoietic cell therapy. Simultaneously, fascinating insights into gene networks that regulate cell fitness will be generated, with important consequences for the fields of hematology, oncology and regenerative medicine.     

Salbutamol versus atropine. Site of bronchodilatation in asthmatic patients

The site of bronchodilatation produced by inhaled salbutamol (0.045 mg/kg) versus atropine (0.035 mg/kg) was investigated in 14 asthmatic young patients. No significant difference was found between the effects of the drugs on any of the large- or small-airway tests in those patients in whom bronchodilatation originated mainly in the large airways or when all patients were considered together. In those patients, however, in whom the small airways made the major contribution to the total airways resistance, atropine produced a higher increase in specific conductance, in maximal flow at all lung volumes and in density dependence of flow, but only the specific conductance showed a significant difference between the effects of the two drugs (p less than 0.01). It was concluded that in these patients atropine produced more bronchodilatation than salbutamol in both central and peripheral airways. These findings do not confirm previous studies which suggested that in asthmatic patients, as in normal subjects, atropine produces mainly central bronchodilatation, while salbutamol produces mainly peripheral bronchodilatation. The difference in doses, route of administration, time response and in the pretreatment severity and site of bronchodilatation could account for the disagreement between previous studies and the present one.     

Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1)

Retroviral vectors are currently the most important and best characterized tools for ex vivo genetic modification of hematopoietic progenitor/stem cells. As a prerequisite for clinical applications, large volumes of high-titer vector supernatants have to be generated in compliance with 'GMP' guidelines. This goal can be reached using a carefully selected producer cell clone and a conventional large-scale cell culture system. The retroviral vector SF1m provides efficient expression of the human multidrug resistance 1 (MDR1) gene in hematopoietic progenitor/stem cells in vitro and in NOD/SCID mouse repopulating human cells in vivo. Currently, a clinical phase I/II study is in preparation to test whether intensified consolidation chemotherapy is enabled by autologous transplantation of peripheral blood progenitor/stem cells that have been genetically modified with SF1m. Using multi-tray cell factories >19 l of serum-free vector containing supernatant were generated from cells of a previously established SF1m-producer clone, based on the PG13 packaging cell line. Testing of the final samples revealed sufficient quality (>1.5 x 10(6) infectious particles/ml) for clinical scale transduction of CD34+ cells. Results from the production runs and the applied biosafety concept are described.     

Correlation of drug sensitivity in vitro with clinical responses in childhood acute myeloid leukemia

Clonogenic cells from 41 children with newly diagnosed acute myeloid leukemia (AML) were tested in vitro for their sensitivity to cytarabine (Ara-C) and daunorubicin (DNR). The findings were then compared with the patients' responses to induction chemotherapy that uniformly included Ara-C and DNR. Light-density marrow cells were incubated with either or both drugs for one hour and cultured over leukocyte feeder layers; clusters and colonies were scored on days 7, 10, and 14. Only the percentage of cell kill in the presence of 1.8 mumol/L DNR was significantly associated with responses to induction therapy: median of 45% (range, 0% to 98%) for patients achieving complete remission v 16% (range, 4% to 23%) for nonresponders (P = .007). The relationship between clonogenic cell kill less than or equal to 23% and clinical responses was striking. Of the 11 evaluable patients with in vitro findings in this category, ten either failed induction therapy or relapsed within 1 year after attaining remission. Kaplan-Meier analysis of relapse-free survival times indicated longer durations of remission for patients whose blast cells showed increased sensitivity in vitro to Ara-C alone, DNR alone, or a combination of the two agents. Seven of 11 patients with cell kills of greater than or equal to 49% in the presence of 1.25 mumol/L Ara-C remain free of leukemia, compared with only one of 12 whose cells were less sensitive to the drug (P = .006). We conclude that the in vitro sensitivity of clonogenic leukemic progenitors to DNR and Ara-C correlates with treatment outcome in children with newly diagnosed AML.     

Prognosis of patients with adenocarcinoma of the lung and mediastinal lymph node metastases undergoing pulmonary resection

Of 57 patients who were operated on for adenocarcinoma of the lung during the period 1966-1970, 18 with mediastinal lymph node metastases successfully underwent potentially curative pulmonary resection combined with complete mediastinal lymph node dissection. The 5-year survival rate was nil. In light of this poor outlook, we do not recommend surgery as the primary treatment of choice in patients with adenocarcinoma of the lung and known mediastinal lymph node metastases.     

Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis

Cell division plays an important role in animal tissue morphogenesis, which depends, critically, on the orientation of divisions. In isolated adherent cells, the orientation of mitotic spindles is sensitive to interphase cell shape and the direction of extrinsic mechanical forces. In epithelia, the relative importance of these two factors is challenging to assess. To do this, we used suspended monolayers devoid of ECM, where divisions become oriented following a stretch, allowing the regulation and function of epithelial division orientation in stress relaxation to be characterized. Using this system, we found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. Nevertheless, because the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. Further, this behavior could be quantitatively reproduced using a model designed to assess the impact of autonomous changes in mitotic cell mechanics within a stretched monolayer. In summary, the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.The morphogenesis of animal tissues results from coordinated changes in the shape, size, and packing of their constituent cells (1–3). These include autonomous cell shape changes (4), the response of cells to extrinsic stresses, and the effects of passive tissue deformation (5). When coordinated across a tissue, these active cellular processes and passive responses enable epithelial sheets to undergo shape changes while retaining relatively normal cell packing (6) and help return tissues to their resting state following a perturbation (7). Although the molecular basis of this cooperation is not understood, several studies have suggested a role for mechanical feedback (8, 9). Cell division has been suggested to participate in this feedback (10) because the rate of animal cell proliferation responds to changes in extrinsic forces in several experimental settings (9). Further, division makes an important contribution to tissue morphogenesis in animals (11, 12), accounts for much of the topological disorder observed in epithelia (13), can drive tissue elongation (10), and can facilitate the return to homeostatic cell packing following a deformation (2). Importantly, for each of these functions, the impact of cell division depends critically on the orientation of divisions.At the cellular level, relatively simple rules appear to govern division orientation. These rules were first explored by Hertwig (14), who showed that cells from early embryos divide along their long axis, and were further refined using microfabricated chambers (15). However, by following division orientation in cells adhering to micropatterned substrates, more recent studies identified additional roles for both the geometrical arrangement of integrin-mediated cell–substrate adhesions (16) and extrinsic mechanical forces in orienting divisions (17). Consistent with this, adhesive and mechanical cues have been reported to guide division orientation in vivo (18) and in epithelial monolayers in developing embryos (12, 19). Nevertheless, the respective roles of cell shape and mechanical tension in guiding division orientation in epithelia remain poorly defined, as does the contribution of oriented division to mechanical feedback control.Previously, we established suspended epithelial monolayers lacking ECM as a minimal model system in which to study epithelial biology. Because cell divisions in these monolayers become oriented following a stretch, we were able to explore the regulation and function of division orientation. We found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. This phenomenon, combined with the alignment of cellular long axes induced by stretch, results in a global bias in the orientation of divisions in the direction of extension. Each division redistributes cell mass along the axis of division. Thus, when oriented across a monolayer, divisions act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. In summary, this analysis shows that the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.