Cobalamin nonresponsive methylmalonic acidemia (MMA, mut complementation class) results from mutations in the nuclear gene MUT, which codes for the mitochondrial enzyme methylmalonyl CoA mutase (MCM). To better elucidate the spectrum of mutations that cause MMA, the MUT gene was sequenced in 160 patients with mut MMA. Sequence analysis identified mutations in 96% of disease alleles. Mutations were found in all coding exons, but predominantly in exons 2, 3, 6, and 11. A total of 116 different mutations, 68 of which were novel, were identified. Of the 116 different mutations, 53% were missense mutations, 22% were deletions, duplications or insertions, 16% were nonsense mutations, and 9% were splice-site mutations. Sixty-one of the mutations have only been identified in one family. A novel mutation in exon 2, c.322C>T (p.R108C), was identified in 16 of 27 Hispanic patients. SNP genotyping data demonstrated that Hispanic patients with this mutation share a common haplotype. Three other mutations were seen exclusively in Hispanic patients: c.280G>A (p.G94R), c.1022dupA, and c.970G>A (p.A324T). Seven mutations were seen almost exclusively in black patients, including the previously reported c.2150G>T (p.G717V) mutation, which was identified in 12 of 29 black patients. Two mutations were seen only in Asian patients. Some frequently identified mutations were not population-specific and were identified in patients of various ethnic backgrounds. Some of these mutations were found in mutation clusters in exons 2, 3, 6, and 11, suggesting a recurrent mutation.

Kucic T, Copland IB, Cuerquis J, Coutu DL, Chalifour LE, Gagnon RF, Galipeau J. Mesenchymal stromal cells genetically engineered to overexpress IGF-I enhance cell-based gene therapy of renal failure-induced anemia.

No abstract

Mesenchymal stromal cells (MSC) are a population of non-hematopoietic progenitors native to the bone marrow that are amenable to genetic engineering, making them attractive delivery vehicles for the in vivo production of therapeutic proteins, such as erythropoietin (Epo). We have previously demonstrated that MSC engineered to secrete Epo can be used for the long-term correction of renal failure-induced anemia [Eliopoulos et al., J Am Soc Nephrol. June 2006]. However, limited long-term transplanted cell survival compromises the efficacy of MSC-based gene therapy approaches. The current study provides evidence that co-implantation of MSC overexpressing Insulin-like growth factor-1 (IGF-I) improves MSC-based gene therapy of anemia by providing paracrine support to Epo-secreting MSC within a synthetic subcutaneous organoid. The IGF-I receptor was found to be expressed in murine MSC by RT-PCR, and protein expression was confirmed by immunoblot. We also demonstrated MSC MAPK pathway responsiveness to IGF-I stimulation in vitro and subsequent improvement of MSC survival following staurosporin-induced apoptosis. Murine MSC were transduced to overexpress either Epo or IGF-I (hereafter MSC-Epo and MSC-IGF) using retroviral vectors. MSC-Epo were subsequently admixed in a collagen matrix and implanted by subcutaneous injection in both naïve mice and a murine model of chronic renal failure, in combination with either MSC-IGF or null MSC. Mice receiving MSC-Epo in conjunction with MSC-IGF experienced a greater and significantly sustained elevation in hematocrit compared to control mice. In addition, mice co-implanted with MSC-IGF and MSC-Epo demonstrated a significant improvement in cardiac function compared to controls. In conclusion, cell-based gene therapy using co-implanted MSC-IGF represents a promising new strategy for the treatment of renal failure-induced anemia, as well as for the improvement of gene-enhanced MSC survival within implanted matrices.