Anemia and Gene Therapy — A Matter of Control
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《新英格兰医药杂志》
All gene-therapy strategies require three elements: a vehicle for gene delivery, a therapeutic gene (sometimes called the transgene), and a physiologically relevant target cell to which the gene is delivered. For some diseases, the choice of target cell is strictly limited — for example, to hematopoietic cells in the case of hemoglobinopathies and to cells in the respiratory tract in the case of the lung disease of cystic fibrosis. For other diseases, such as plasma protein deficiencies (e.g., hemophilia or erythropoietin-responsive anemia), there is latitude in the choice of target cell, as long as the transgene product can be secreted into the circulation. Along these lines is a strategy, recently described by Takács et al.,1 that harnesses the ability of B cells to proliferate in response to a specific antigen and thus allows the amount of the resulting therapeutic gene product to be controlled.
The proof-of-principle experiments that Takács et al. describe (Figure 1) make use of a somewhat elaborate system, but the potential clinical application is, if anything, more straightforward. They began with transgenic mice engineered to express human erythropoietin under the control of a B-cell–specific element. The mice were then immunized with a specific antigen, phycoerythrin (which is unrelated to erythropoietin), giving rise to a pool of B cells that would proliferate and express erythropoietin when stimulated by exposure to phycoerythrin. The mice were then killed, and their splenic and lymphatic lymphocytes were harvested and transferred to mice with anemia due to erythropoietin deficiency. Serum erythropoietin levels and hematocrit values increased in the recipient mice in response to the administration of phycoerythrin, and the time course of this response was consistent with the known kinetics of the clonal expansion of B cells in response to an antigen. Furthermore, the effect was maintained during several cycles of antigen challenge.
Figure 1. Eliciting Erythropoietin.
A means of stimulating the production of erythropoietin and its secretion into the bloodstream could be the basis of a treatment for anemia. Takács et al.1 have recently described a strategy that achieves this goal in a mouse model. They used a genetically modified, antigen-specific B cell to supply erythropoietin to a mouse deficient in erythropoietin. Transgenic mice were engineered to express human erythropoietin under the control of a B-cell–specific element (Panel A). The mouse was then immunized with a single antigen, phycoerythrin, resulting in a pool of antigen-sensitive B cells (Panel B). Lymphocytes from the donor mouse were injected into an erythropoietin-deficient mouse (Panel C). Subsequent injection of antigen into the erythropoietin-deficient mice stimulated the proliferation of B cells expressing erythropoietin, with a resultant increase in serum erythropoietin levels and hematocrit values (Panel D).
How might these findings be applied to the treatment of disease in humans? Instead of using cells from a donor, one would use the patient's own cells. The patient would first be immunized with some harmless antigen to create a pool of B cells responsive to that antigen. Peripheral-blood mononuclear cells could then be obtained from the patient by means of leukapheresis, and antigen-specific cells could be isolated and transduced with a vector expressing the therapeutic gene under the control of a promoter that drives gene expression in activated B cells (such as the one that controlled the expression of erythropoietin in the transgenic mice). The cells would then be returned to the patient. When challenged with antigen, the patient's antigen-specific B cells would be expected to proliferate and secrete the therapeutic gene product. If this approach works in humans as it does in mice, repeated rounds of the antigen could be administered to elevate the levels of therapeutic protein as needed.
Of course, strategies that rely on the expansion of a transfected cell population require an integrating vector, which would expose patients to the risk of insertional mutagenesis. This risk had long been acknowledged but had largely been viewed as theoretical until reports two years ago of two children in whom a leukemia-like syndrome developed after successful gene therapy for X-linked severe combined immunodeficiency.2 In both patients, analysis showed that the vector was inserted into the genome in the vicinity of a gene encoding a protein involved in gene transcription, which may have triggered leukemogenesis. Research efforts have since focused on potential strategies for reducing or eliminating the risk of insertional mutagenesis. Possible solutions include adding a "suicide" gene to the transgene cassette to render transduced cells susceptible to killing by antibiotics,3 using insulating elements to prevent the promoter sequences of the donated gene from influencing or being influenced by neighboring genes,4 developing vectors with a very restricted set of integration sites in the mammalian genome, and perfecting ways to induce site-specific recombination to repair a mutation.5 Although the strategy described by Takács et al.1 is promising, its application depends on the development of such safety measures.
Source Information
From the Howard Hughes Medical Institute, the Children's Hospital of Philadelphia, and the University of Pennsylvania — all in Philadelphia.
References
Takács K, Du Roure C, Nabarro S, et al. The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes. Proc Natl Acad Sci U S A 2004;101:16298-16303.
Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255-256.
Kirn D, Niculescu-Duvaz I, Hallden G, Springer CJ. The emerging fields of suicide gene therapy and virotherapy. Trends Mol Med 2002;8:Suppl 4:S68-S73.
Chung JH, Bell AC, Felsenfeld G. Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci U S A 1997;94:575-580.
Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science 2003;300:763-763.(Katherine High, M.D.)
The proof-of-principle experiments that Takács et al. describe (Figure 1) make use of a somewhat elaborate system, but the potential clinical application is, if anything, more straightforward. They began with transgenic mice engineered to express human erythropoietin under the control of a B-cell–specific element. The mice were then immunized with a specific antigen, phycoerythrin (which is unrelated to erythropoietin), giving rise to a pool of B cells that would proliferate and express erythropoietin when stimulated by exposure to phycoerythrin. The mice were then killed, and their splenic and lymphatic lymphocytes were harvested and transferred to mice with anemia due to erythropoietin deficiency. Serum erythropoietin levels and hematocrit values increased in the recipient mice in response to the administration of phycoerythrin, and the time course of this response was consistent with the known kinetics of the clonal expansion of B cells in response to an antigen. Furthermore, the effect was maintained during several cycles of antigen challenge.
Figure 1. Eliciting Erythropoietin.
A means of stimulating the production of erythropoietin and its secretion into the bloodstream could be the basis of a treatment for anemia. Takács et al.1 have recently described a strategy that achieves this goal in a mouse model. They used a genetically modified, antigen-specific B cell to supply erythropoietin to a mouse deficient in erythropoietin. Transgenic mice were engineered to express human erythropoietin under the control of a B-cell–specific element (Panel A). The mouse was then immunized with a single antigen, phycoerythrin, resulting in a pool of antigen-sensitive B cells (Panel B). Lymphocytes from the donor mouse were injected into an erythropoietin-deficient mouse (Panel C). Subsequent injection of antigen into the erythropoietin-deficient mice stimulated the proliferation of B cells expressing erythropoietin, with a resultant increase in serum erythropoietin levels and hematocrit values (Panel D).
How might these findings be applied to the treatment of disease in humans? Instead of using cells from a donor, one would use the patient's own cells. The patient would first be immunized with some harmless antigen to create a pool of B cells responsive to that antigen. Peripheral-blood mononuclear cells could then be obtained from the patient by means of leukapheresis, and antigen-specific cells could be isolated and transduced with a vector expressing the therapeutic gene under the control of a promoter that drives gene expression in activated B cells (such as the one that controlled the expression of erythropoietin in the transgenic mice). The cells would then be returned to the patient. When challenged with antigen, the patient's antigen-specific B cells would be expected to proliferate and secrete the therapeutic gene product. If this approach works in humans as it does in mice, repeated rounds of the antigen could be administered to elevate the levels of therapeutic protein as needed.
Of course, strategies that rely on the expansion of a transfected cell population require an integrating vector, which would expose patients to the risk of insertional mutagenesis. This risk had long been acknowledged but had largely been viewed as theoretical until reports two years ago of two children in whom a leukemia-like syndrome developed after successful gene therapy for X-linked severe combined immunodeficiency.2 In both patients, analysis showed that the vector was inserted into the genome in the vicinity of a gene encoding a protein involved in gene transcription, which may have triggered leukemogenesis. Research efforts have since focused on potential strategies for reducing or eliminating the risk of insertional mutagenesis. Possible solutions include adding a "suicide" gene to the transgene cassette to render transduced cells susceptible to killing by antibiotics,3 using insulating elements to prevent the promoter sequences of the donated gene from influencing or being influenced by neighboring genes,4 developing vectors with a very restricted set of integration sites in the mammalian genome, and perfecting ways to induce site-specific recombination to repair a mutation.5 Although the strategy described by Takács et al.1 is promising, its application depends on the development of such safety measures.
Source Information
From the Howard Hughes Medical Institute, the Children's Hospital of Philadelphia, and the University of Pennsylvania — all in Philadelphia.
References
Takács K, Du Roure C, Nabarro S, et al. The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes. Proc Natl Acad Sci U S A 2004;101:16298-16303.
Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255-256.
Kirn D, Niculescu-Duvaz I, Hallden G, Springer CJ. The emerging fields of suicide gene therapy and virotherapy. Trends Mol Med 2002;8:Suppl 4:S68-S73.
Chung JH, Bell AC, Felsenfeld G. Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci U S A 1997;94:575-580.
Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science 2003;300:763-763.(Katherine High, M.D.)