Preclinical studies for a phase 1 clinical trial of autologous hematopoietic stem cell gene therapy for sickle cell disease.

Background Aims: Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials.

Methods: Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/β-FB vector and cryopreserving CD34 cells from human bone marrow (BM) at clinical scale.

Molecular, phenotypic, and sample-associated data to describe pluripotent stem cell lines and derivatives.

The use of induced pluripotent stem cells (iPSC) derived from independent patients and sources holds considerable promise to improve the understanding of development and disease. However, optimized use of iPSC depends on our ability to develop methods to efficiently qualify cell lines and protocols, monitor genetic stability, and evaluate self-renewal and differentiation potential. To accomplish these goals, 57 stem cell lines from 10 laboratories were differentiated to 7 different states, resulting in 248 analyzed samples.

Overcoming reprogramming resistance of Fanconi anemia cells.

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction.

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway.

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20).

Interlaboratory assessment of a novel colony-forming unit assay: a multicenter study by the cellular team of Biomedical Excellence for Safer Transfusion (BEST) collaborative.

Background: Interlaboratory scoring performances were determined using a traditional 14-day colony-forming unit (CFU) assay and a new 7-day CFU assay.

Study Design And Methods: Digital images of colonies were utilized to train personnel at each site. A central laboratory inoculated methylcellulose with progenitors and sent the samples by overnight courier to participating labs for plating.

Correct mRNA processing at a mutant TT splice donor in FANCC ameliorates the clinical phenotype in patients and is enhanced by delivery of suppressor U1 snRNAs.

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees.

Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M.

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated.

Database setup for preclinical studies of gene-modified hematopoiesis.

Murine safety studies are routinely used for gathering preclinical safety and efficacy data and, for Phase I studies, Good Laboratory Practice (GLP) compliance is not mandated. However, extensive amounts of data must be gathered and analyzed. An inter-relational database is an effective tool for storing, sorting, and reviewing data.

Copy number determination of genetically-modified hematopoietic stem cells.

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome.

Physiological promoters reduce the genotoxic risk of integrating gene vectors.

The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer-promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1alpha (EF1alpha) and phosphoglycerate kinase (PGK).

Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency.

Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1.

Insertional mutagenesis by replication-deficient retroviral vectors encoding the large T oncogene.

Insertion sites of replication-deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor-suppressor proteins p53 and Rb by virtue of a chaperone-like activity.