Targeting Gene Therapy for Osteogenesis Imperfecta
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《新英格兰医药杂志》
Two shadows have recently been cast over the exciting field of gene therapy. One was cast by the unfortunate death of an 18-year-old man at the University of Pennsylvania in 1999.1 The patient was part of a trial in which an adenovirus containing a gene for ornithine transcarbamylase was infused into patients with partial deficiencies of the enzyme. The death highlighted the potential dangers of the direct administration of adenoviruses and, perhaps, other viruses to patients. The second shadow was cast by a clinical trial involving children with X-linked severe combined immunodeficiency (SCID).2 The trial had an added measure of safety in that the genetic defect was first corrected in lymphocytes from the patients by using a replication-deficient retrovirus ex vivo; the modified cells were then infused into the patients. The initial results were hailed as the first instance of successful gene therapy. Unfortunately, lymphocytic leukemia has since developed in two of the nine patients.2 The retrovirus inserted itself near a proto-oncogene and thereby triggered premalignant proliferation of the cells in these patients, in whom there was a strong selection for rapidly proliferating lymphocytes. Until the SCID trial, the unfortunate effects of insertional mutagenesis seemed more a theoretical than a practical danger, since it had rarely, if ever, been seen in hundreds of experiments in animals.
Chamberlain et al.3 recently reported a clever first step in minimizing the dangers of insertional mutagenesis. They focused on osteogenesis imperfecta, in which bones are fragile because of mutations in either of the two genes that encode subunits of type I collagen. In lethal forms of the disease, the mutations result in the synthesis of abnormal chains of procollagen that bind to normal chains synthesized by the same cells and destroy their biologic activity in a classic dominant negative manner. Chamberlain et al. developed a strategy to inactivate the mutated alleles in cells of the bone marrow called mesenchymal stem cells (MSCs), or marrow stromal cells. They chose MSCs because these cells are easily obtained from a patient, they engraft and differentiate into many tissues after infusion in vivo, and allogeneic MSCs had produced promising results in a previous trial involving patients with osteogenesis imperfecta.4,5
Chamberlain et al. designed a gene construct that targets exon 1 of the gene for collagen type I1 (COL1A1), which encodes one of the two collagen subunits. They predicted that, on insertion, the construct would both inactivate COL1A1 and confer resistance to the antibiotic neomycin (Figure 1). To insert the gene construct efficiently into MSCs, they used an adenoassociated virus as a vector, which, unlike adenoviruses, is integrated into chromosomal sites.
Figure 1. Boning Up on Gene Therapy.
Type I collagen, a structural constituent of bone, is made up of two subunits, one of which is encoded by the COL1A1 gene (Panel A). Mutations in the genes encoding these subunits cause osteogenesis imperfecta. Dominant negative mutations result in mutant protein that interferes with the activity of wild-type protein encoded by the unaffected gene (Panel B). Diminishing the levels of mutant protein may be a way to treat the disease, and a recent study by Chamberlain et al.3 provides support for this approach and suggests that it could be achieved through gene therapy. Using a molecular construct engineered to insert itself into the COL1A1 gene (Panel C), the investigators showed that the processing and stability of collagen could be improved by the targeting of mutant mesenchymal stem cells and that the targeted cells become bone cells. Although the construct probably targets mutated and wild-type alleles equally, the approach works because the cells in which the wild-type allele is inactivated do not produce collagen and the cells in which the mutated allele is targeted produce adequate amounts of the normal protein.
The results obtained with MSCs from two patients with osteogenesis imperfecta were extremely encouraging. In 31 to 90 percent of the cells that became resistant to neomycin, the gene construct had inserted itself into either the wild-type or the mutated COL1A1 allele. In all cultures of the neomycin-resistant cells, most signs of the dominant negative protein defect were corrected — apparently because the cells in which the mutated allele was inactivated began to produce an adequate amount of wild-type collagen. Most important, the quality of bone synthesized by the altered MSCs was improved, as evidenced by the results of a standard assay in which the MSCs were infused into ceramic cubes and the cubes were then implanted into immunodeficient mice.
Precise targeting of exogenous genes to replace mutated genes has long been the holy grail of gene therapy. Gene targeting by homologous recombination occurs with very high frequency in yeast, but until now, it has not been possible to increase the very low frequency of this event in mammalian cells. The results of the study by Chamberlain et al. clearly take us a step closer to targeting exogenous genes in ex vivo gene therapy. Their strategy for correcting the dominant negative protein defects in osteogenesis imperfecta can probably be adapted for use in other genetic defects, particularly those involving small genes that can readily be accommodated within a viral vector.
However, a few problems remain. One is that the neomycin-resistance gene that was used predisposed the cells for destruction, apparently because the foreign protein invokes immune responses.5 Another problem is that although the gene was correctly targeted in 31 to 90 percent of the neomycin-resistant MSCs — a dramatic improvement over previous efforts involving human cells — insertional mutagenesis is still a possibility in the remaining cells. Both these problems could be solved by measures such as the use of different markers and more extensive selection of the cells. In the end, gene therapy will work, given the large cadre of talented scientists addressing the problems and the number of tools for molecular and cellular biology now at our disposal.
Source Information
From the Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans.
References
Raper SE, Chirmule N, Lee FS, et al. Fatal systemic inflammatory response syndrome in an ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 2003;80:148-158.
McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2004;350:913-922.
Chamberlain JR, Schwarze U, Wang P-R, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198-1201.
Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A 2003;100:Suppl 1:11917-11923.
Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A 2002;99:8932-8937.(Darwin J. Prockop, M.D., )
Chamberlain et al.3 recently reported a clever first step in minimizing the dangers of insertional mutagenesis. They focused on osteogenesis imperfecta, in which bones are fragile because of mutations in either of the two genes that encode subunits of type I collagen. In lethal forms of the disease, the mutations result in the synthesis of abnormal chains of procollagen that bind to normal chains synthesized by the same cells and destroy their biologic activity in a classic dominant negative manner. Chamberlain et al. developed a strategy to inactivate the mutated alleles in cells of the bone marrow called mesenchymal stem cells (MSCs), or marrow stromal cells. They chose MSCs because these cells are easily obtained from a patient, they engraft and differentiate into many tissues after infusion in vivo, and allogeneic MSCs had produced promising results in a previous trial involving patients with osteogenesis imperfecta.4,5
Chamberlain et al. designed a gene construct that targets exon 1 of the gene for collagen type I1 (COL1A1), which encodes one of the two collagen subunits. They predicted that, on insertion, the construct would both inactivate COL1A1 and confer resistance to the antibiotic neomycin (Figure 1). To insert the gene construct efficiently into MSCs, they used an adenoassociated virus as a vector, which, unlike adenoviruses, is integrated into chromosomal sites.
Figure 1. Boning Up on Gene Therapy.
Type I collagen, a structural constituent of bone, is made up of two subunits, one of which is encoded by the COL1A1 gene (Panel A). Mutations in the genes encoding these subunits cause osteogenesis imperfecta. Dominant negative mutations result in mutant protein that interferes with the activity of wild-type protein encoded by the unaffected gene (Panel B). Diminishing the levels of mutant protein may be a way to treat the disease, and a recent study by Chamberlain et al.3 provides support for this approach and suggests that it could be achieved through gene therapy. Using a molecular construct engineered to insert itself into the COL1A1 gene (Panel C), the investigators showed that the processing and stability of collagen could be improved by the targeting of mutant mesenchymal stem cells and that the targeted cells become bone cells. Although the construct probably targets mutated and wild-type alleles equally, the approach works because the cells in which the wild-type allele is inactivated do not produce collagen and the cells in which the mutated allele is targeted produce adequate amounts of the normal protein.
The results obtained with MSCs from two patients with osteogenesis imperfecta were extremely encouraging. In 31 to 90 percent of the cells that became resistant to neomycin, the gene construct had inserted itself into either the wild-type or the mutated COL1A1 allele. In all cultures of the neomycin-resistant cells, most signs of the dominant negative protein defect were corrected — apparently because the cells in which the mutated allele was inactivated began to produce an adequate amount of wild-type collagen. Most important, the quality of bone synthesized by the altered MSCs was improved, as evidenced by the results of a standard assay in which the MSCs were infused into ceramic cubes and the cubes were then implanted into immunodeficient mice.
Precise targeting of exogenous genes to replace mutated genes has long been the holy grail of gene therapy. Gene targeting by homologous recombination occurs with very high frequency in yeast, but until now, it has not been possible to increase the very low frequency of this event in mammalian cells. The results of the study by Chamberlain et al. clearly take us a step closer to targeting exogenous genes in ex vivo gene therapy. Their strategy for correcting the dominant negative protein defects in osteogenesis imperfecta can probably be adapted for use in other genetic defects, particularly those involving small genes that can readily be accommodated within a viral vector.
However, a few problems remain. One is that the neomycin-resistance gene that was used predisposed the cells for destruction, apparently because the foreign protein invokes immune responses.5 Another problem is that although the gene was correctly targeted in 31 to 90 percent of the neomycin-resistant MSCs — a dramatic improvement over previous efforts involving human cells — insertional mutagenesis is still a possibility in the remaining cells. Both these problems could be solved by measures such as the use of different markers and more extensive selection of the cells. In the end, gene therapy will work, given the large cadre of talented scientists addressing the problems and the number of tools for molecular and cellular biology now at our disposal.
Source Information
From the Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans.
References
Raper SE, Chirmule N, Lee FS, et al. Fatal systemic inflammatory response syndrome in an ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 2003;80:148-158.
McCormack MP, Rabbitts TH. Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2004;350:913-922.
Chamberlain JR, Schwarze U, Wang P-R, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198-1201.
Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A 2003;100:Suppl 1:11917-11923.
Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A 2002;99:8932-8937.(Darwin J. Prockop, M.D., )