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.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.