568. Characterization of Iduronidase-Expressing Monocyte Progenitor Cells for a Cell Based Therapy for Hurler Syndrome
Authors: Simon Lee, Saul Kivimäe, Francis C. Szoka
Journal: Molecular Therapy (2015)

Macrophages (MFs) possess unique homing characteristics into tumors and sites of inflammation. They can also repopulate niches that have been depleted of endogenous MFs, which may be useful for the development of novel gene therapies for lysosomal storage disorders. We have implemented methods to reliably produce and engineer MFs using a lentivirus to transduce bone marrow derived myeloid progenitors (MPs) to conditionally express Hoxb8 at high levels under the control of CreERT. This allows MPs to differentiate into proliferative monocyte progenitors (MoPs), but prevents further differentiation into non-proliferative MFs. Simultaneous incorporation of multiple orthogonal lox and flippase recognition target (FRT) sites during the lentiviral transduction and subsequent transfection allows for insertion of therapeutic or reporter genes. This is accomplished by either activating CreERT to excise sequences flanked by lox sites, or co-transfecting flippase with plasmids containing FRT sites matching those incorporated in the genome, allowing for recombinase-mediated cassette exchange to occur and the introduction of new functional genes. Addition of 10nM 4-hydroxytamoxifen results in activation of CreERT and the excision of Hoxb8, allowing MoPs to differentiate into MFs over two weeks. By first genetically engineering the cells in the MoP state, and then inducing MΦ differentiation, this strategy enables us to generate modified MFs with a range of characteristics in a highly controlled manner for use in cell based gene therapies. Characterization of these cells shows that MoPs are highly proliferative, doubling every 12h in suspension culture. Furthermore, MoPs grown in hypoxic environments, ranging from 2.5%-18% O2, similar to those observed in tissue and inflammatory sites, showed little variation in growth or behavior. Thus, these cells represent a viable option for long-term implantation in macrophage niches, including the liver and spleen. Hurler syndrome is a lysosomal storage disease caused by the inability to generate functional iduronidase (IDUA), an enzyme critical for the degradation of heparan sulfate, a key component of the extracellular matrix. Accumulation of heparan sulfate leads to significant cardiovascular, skeletal and neural defects. Restoration of IDUA, through bone marrow transplant from an individual with functional IDUA or enzyme replacement therapy, is currently the method of choice for treatment, but represent significant transplantation risks or lack long-term efficacy. A cell based therapy, where MFs expressing IDUA are transplanted into MΦ niches would reduce risks while providing long-term efficacy. Transfecting IDUA into Cos7 cells has shown functional IDUA is secreted, with activity 1000x above endogenous levels. Results and characterization of engineered Mo/MFs to express functional IDUA will be presented. These cells, following depletion of endogenous MFs with liposomal clodronate, can be transplanted and repopulate niches in the liver and spleen, creating a long-lived depot of IDUA-secreting cells and allowing for systemic, long-term delivery of IDUA. Supported by NIH R01EB003008 and a NSERC-PGSD fellowship to Simon Lee.