Overnight transduction with foamyviral vectors restores the long-term repopulating activity of Fancc-/- stem cells.

Fanconi anemia (FA) is a complex genetic disorder characterized by congenital abnormalities, bone marrow failure, and myeloid malignancies. Identification of 13 FA genes has been instrumental to explore gene transfer technologies aimed at correction of autologous FA-deficient stem cells. To date, 3 human FA stem cell gene therapy trials with standard 4-day transduction protocols using gammaretroviral vectors failed to provide clinical benefit. In addition, 2- to 4 day ex vivo manipulation of bone marrow from mice containing a disruption of the homologue of human FANCC (Fancc) results in a time-dependent increase in apoptosis and a risk for malignant transformation of hematopoietic cells. Here, we show that a 14-hour transduction period allows a foamyviral vector construct expressing the human FANCC cDNA to efficiently transduce murine FA stem cells with 1 to 2 proviral integrations per genome. Functionally, the repopulating activity of Fancc(-/-) stem cells from reconstituted mice expressing the recombinant FANCC transgene was comparable with wild-type controls. Collectively, these data provide evidence that short-term transduction of c-kit(+) cells with a foamyviral vector is sufficient for functional correction of a stem cell phenotype in a murine FA model. These data could have implications for future gene therapy trials for FA patients.

In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMT(P140K) vector.

OBJECTIVE: Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin(neg) BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and selected in vivo with submyeloablative or myeloablative alkylator therapy. MATERIALS AND METHODS: Lin(neg) BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT(P140K) and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals. RESULTS: Following submyeloablative therapy, 55% of the mice expressed MGMT(P140K) in the BM. Proviral integration was observed in approximately 50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT(P140K)-positive cells for 5 to 6 months. CONCLUSIONS: In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative than myeloablative therapy. These data indicate that a critical number of transduced stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against acute toxicity associated with myeloablative therapy.

Identification of TEL-AML1 (ETV6-RUNX1) associated DNA and its impact on mRNA and protein output using ChIP, mRNA expression arrays and SILAC.

The contribution of the most common reciprocal translocation in childhood B-cell precursor leukemia t(12;21)(p13;q22) to leukemia development is still under debate. Direct as well as secondary indirect effects of the TEL-AML1 fusion protein are commonly recorded by using cell lines and patient samples, often bearing the TEL-AML1 fusion protein for decades. To identify direct targets of the fusion protein a short-term induction of TEL-AML1 is needed. We here describe in detail the experimental procedure, quality controls and contents of the ChIP, mRNA expression and SILAC datasets associated with the study published by Linka and colleagues in the Blood Cancer Journal [1] utilizing a short term induction of TEL-AML1 in an inducible precursor B-cell line model.

Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype.

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.