ALBERT

Description: Abstract 3584 Poster Board III-521 Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A (α-gal A) activity that results in the widespread accumulation of neutral glycosphingolipids. Renal failure, neuropathy, premature myocardial infarction, and stroke occur in patients with this condition due to deposition of globotriaosylceramide (Gb3) in vascular endothelial cells. Gb3 primarily originates from the breakdown of red blood cells. Currently, Fabry disease is mainly treated by enzyme replacement therapy (ERT). This treatment represents only a short-term therapeutic strategy and does not solve the basic genetic defect of the disorder. Fabry patients have also shown variable and muted response to ERT. Genetically-modified hematopoietic stem cells (HSCs) are attractive targets for durable transgene expression, however lower transduction efficiencies may be an obstacle to attainment of successful therapy. Therefore, our goal is to design and implement a recombinant lentiviral vector (LV) to confer drug tolerance to modified Fabry patient HSCs in order to enrich them in vivo. O6-methylguanine-DNA-methyltransferase (MGMT) overexpression provides cellular resistance to alkylating agents, which can be administered to kill residual untransduced HSCs while modified cells are protected. To this end, we have developed two different LV constructs. The first iteration was represented by a bicistronic LV encoding the human α-gal A cDNA followed by an EMCV-IRES element and a mutated, but functional, form of the selectable marker (MGMT-P140K). Next, a second iteration has been developed with the order of the transgenes reversed and carrying a codon-optimized copy of the human α-gal A cDNA. Expression and functionality of both cassettes were tested in the human erythroleukaemic cell line (K562) by intracellular MGMT staining and α-gal A activity assays, before and after selection with O6-benzylguanine (BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We found that the second construct allowed sufficient MGMT expression for selection of transduced cells while maintaining α-gal A expression at appreciable levels. Flow cytometry analysis revealed that one drug treatment was sufficient to increase the MGMT-positive cell population from 15% to 90%, which resulted in a 2-fold increase in the enzyme activity. However, two treatments were required for achieving a similar result with the LV of the first iteration. The second LV also facilitated enrichment of transduced Fabry B cells in vitro. Future directions on this project include the modification of Fabry patient bone marrow cells, transplantation into a novel xenograft mouse model that we have recently created, and selection and amplification of the modified cells in vivo. Considering that clinical evidence suggests that even a slight increase in α-gal A activity may help to correct the disease, these results suggest that the MGMT-based drug selection system holds promise for providing a cure for Fabry Disease with a single gene therapy treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

Description: Abstract 3576 Poster Board III-513 Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A (α-gal A). The inability to prevent the progression of galactosylsphingolipid deposition, such as globotriaosylceramide (Gb3), has a significant impact on quality of life and diminishes lifespan from early-onset strokes, progressive renal failure, and heart attacks. Previously, we have demonstrated that gene transfer into murine hematopoietic cells can correct the defect systemically in Fabry mice. The goal of the present study is to create a pure Fabry/NOD/SCID murine line to facilitate the in vivo assessment of human cell-targeted therapies against the disease. The pure line was generated by a “speed congenic” breeding program. The parental generation (F0) was represented by a C3H+C57BL/6 Fabry female mouse (α-gal A−/− scid+/+) and a NOD/SCID male mouse (α-gal A+/0 scid−/−). To generate the α-gal A-/+ scid−/− female mice (F2), the double heterozygous female mice from F1 were mated with NOD/SCID male mice. F3 and all the subsequent generations (until F11) were derived by backcrossing α-gal A-/+ scid−/− female mice with α-gal A+/0 scid−/− male mice. At this point, genome scanning analysis, fluorometric enzymatic assays, and HPLC assessment revealed that the F11 purity was higher than 99%; α-gal A activity was reduced significantly in plasma and Gb3 levels were increased considerably in heart, liver, spleen, kidney and lung, in comparison to NOD/SCID control mice. With the aim of obtaining the pure Fabry/NOD/SCID line, F11 mice were crossed with each other (α-gal A-/+ scid−/− female mice with α-gal A-/0 scid−/− male mice) and then F12 double homozygous female mice (α-gal A−/− scid−/−) were crossed with F12 hemizygous (α-gal A-/0 scid−/−) male mice. For each generation, the genotype of offspring was analyzed by PCR and the absence of T and B cells was confirmed phenotypically by flow cytometry. Currently, this new xenograft model is being validated by using hematopoietic cell targets. To this end, normal human mobilized CD34+ cells were separately transduced with a control (eGFP lentivector) or a bicistronic lentiviral vector encoding the human α-gal A and the human CD25 cell surface marker. 8×105 cells were injected intravenously into sub-lethally irradiated 8-week-old pure Fabry/NOD/SCID male mice. Injected cells were 25% positive for eGFP and 34% positive for human CD25 expression, respectively. Presence of human CD45+ cells, CD45+/CD25+ cells, CD45+/eGFP+ cells and α-gal A activity will be regularly monitored on peripheral blood or plasma, respectively. Near future studies will include Gb3 quantification in tissues at sacrifice and secondary recipient transplantation. As well, Fabry patient bone marrow cells are currently being collected under an approved protocol for testing in this model. In conclusion, this novel xenograft Fabry model is a key tool for developing different therapies for Fabry Disease and will help us to reduce the gap between the bench and the clinic. Disclosures: No relevant conflicts of interest to declare.