Novel Treatment for Osteogenesis Imperfecta
© 2004 Prime Health Consultants, Inc.
Osteogenesis imperfecta (OI) is a relatively common genetic disorder characterized by bone fragility, skeletal deformities, ligamentous laxity, thin skin, blue sclerae, and other “connective tissue” features. It results from heterozygous mutations of the COL1A1 and COL1A2 genes that encode the pro 1 and pro a 2 chains of type I collagen; its manifestations reflect the distribution of type I collagen. The most severe forms of OI, eg, OI type II, result from mutations in which the mutant procollagen chains are synthesized, participate in and disrupt the assembly of triple helical type I collagen molecules. Such mutant genes or alleles might be considered dysfunctional alleles. In contrast, milder forms, such as OI type I, result from mutations that inactivate one of the two COL1A1 or COL1A2 alleles, leaving the patient with only one functional COL1A1 or COL1A2 allele. Accordingly, one strategy to treat severe OI is to convert the dysfunctional COL1 allele to a nonfunctional or null allele. A group headed by David Russell (Chamberlain et al) has demonstrated the feasibility of inactivating dysfunctional COL1A1 alleles in adult, or often termed mesenchymal, stem cells (MSC) from patients with severe OI.
MSC available from the marrow of bone samples discarded at surgery, have the potential to repopulate bone marrow and locally generate new bone. In principle, MSC could be harvested from a patient with severe OI, genetically manipulated to inactivate a dysfunctional type I collagen gene, and be transplanted back into the patient where they would home to bone marrow and locally produce bone tissue with properties of mild OI.
In this investigation, MSC were isolated from 2 patients with severe OI whose specific mutations were identified and shown to be of the dysfunctional type. The cells were infected with an adeno-associated virus-based gene targeting vector designed to promote homologous recombination between the vector and the COL1A1 gene. Successful targeting inserted foreign DNA, including the gene for neomycin resistance, into exon 1 of the COL1A1 gene, thereby preventing its expression and converting it to a null allele. The targeting vector did not distinguish between the mutant and normal COL1A1 allele. The MSC were then grown in an antibiotic to select recombinant cells and then analyzed to confirm that targeting was successful. Analysis of several cell clones, as well as pools of cells, revealed that a high percentage of the resistant MSC had undergone targeting at one COL1A1 allele. Significant improvements were observed in measures of collagen processing, stability, and fibril ultrastructure for targeted cells, and accumulation of suspected mutant collagen present in the original cells disappeared in the targeted MSC. To determine if the targeted MSC retained their ability to become osteoblasts, the targeted MSC were implanted subcutaneously into immunodeficient mice, removed after 8 weeks, and analyzed. Although amount varied, bone formation was detected in all of the implants and the osteocytes were shown to be of human origin.
The authors note potential problems with their approach, such as inability to specifically target the mutant versus the normal COL1A1 allele and possible immunogenicity of the foreign neomycin-resistance gene. But they argue that these can eventually be overcome and that their approach has certain advantages over allogeneic bone marrow and MSC transplantation, which has been reported in a clinical trial for severe OI.
First Editor’s Comment: There are 2 major challenges in order for this form of genetic treatment to be successful. The first is being able to alter the mutant gene so that the mutation is corrected or nullified, as in this case. This paper demonstrates that conversion of a dominant-negatively acting allele to a null allele works, at least in cell culture and in mice, and can be carried out in a time frame that is realistic for clinical use. Although there are still many problems to resolve, the gene-targeting strategy has considerable promise. The second challenge is to achieve sufficient engraftment of genetically modified cells to repair excessively fragile bones. Fortunately, therapists can exploit the high vascularity of bone and the natural behavior of MSC to home to marrow and differentiate as functional osteoblasts. However, previous attempts at allogeneic MSC transplantation and similar experiments in mice have resulted in modest engraftment, at best. Figuring out how to safely and effectively impart therapeutic cells with a competitive advantage over their dysfunctional endogenous counterparts in bone may prove to be the greater of the 2 challenges. Nevertheless, given the absence of other successful treatments for severe OI, it remains a viable potential option.
William A. Horton, MD
Second Editor’s Comment: The exciting field of gene therapy has been given a shot in the arm by these studies The recent comment by Prockop1 on targeting gene therapy for OI is worth reading in order to facilitate the appreciation of this novel concept (figure).
Fima Lifshitz, MD
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