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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.
Chamberlain JR, Schwarze U, Wang PR, Hirata RK, Hankenson KD, Pace JM, Underwood RA, Song KM, Sussman M, Byers PH, Russell DW.Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198-1201.
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
Reference - (linked to  )
- Prockop DJ. Targeting gene therapy for osteogenesis imperfecta. N Engl J Med 2004;350:2302-234.
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Current GGH - Vol. 24 No. 1
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