Search

Use of megakaryocytes/platelets for transgene expression may take advantage of their rapid turnover and protective storage in platelets and reduce the risk of activating oncogenes in hematopoietic stem and progenitor cells (HSCs). Here, we show that human megakaryocytic cells could overexpress the lysosomal enzyme, α-l-iduronidase (IDUA), which is deficient in patients with mucopolysaccharidosis type I (MPS I). Upon megakaryocytic differentiation, the amount of released enzyme increased rapidly and steadily by 30-fold. Using a murine MPS I model, we demonstrated that megakaryocyte/platelets were capable of producing, packaging, and storing large amounts of IDUA with proper catalytic activity, lysosomal trafficking, and receptor-mediated uptake. IDUA can be released directly into extracellular space or within microparticles during megakaryocyte maturation or platelet activation, while retaining the capacity for cross-correction in patient’s cells. Gene transfer into 1.7% of HSCs led to long-term normalization of plasma IDUA and preferential distribution of enzyme in liver and spleen with complete metabolic correction in MPS I mice. Detection of GFP (coexpressed with IDUA) in Kupffer cells and hepatocytes suggested liver delivery of platelet-derived IDUA possibly via the clearance pathway for senile platelets. These findings provide proof of concept that cells from megakaryocytic lineage and platelets are capable of generating and storing fully functional lysosomal enzymes and can also lead to efficient delivery of both the enzymes released into the circulation and those protected within platelets/microparticles. This study opens a door for use of the megakaryocytes/platelets as a depot for efficient production, delivery, and effective tissue distribution of lysosomal enzymes.

To realize the potential of large molecular weight substances to treat neurological disorders, novel approaches are required to surmount the blood–brain barrier (BBB). We investigated whether fusion of a receptor-binding peptide from apolipoprotein E (apoE) with a potentially therapeutic protein can bind to LDL receptors on the BBB and be transcytosed into the CNS. A lysosomal enzyme, α- L -iduronidase (IDUA), was used for biological and therapeutic evaluation in a mouse model of mucopolysaccharidosis (MPS) type I, one of the most common lysosomal storage disorders with CNS deficits. We identified two fusion candidates, IDUAe1 and IDUAe2, by in vitro screening, that exhibited desirable receptor-mediated binding, endocytosis, and transendothelial transport as well as appropriate lysosomal enzyme trafficking and biological function. Robust peripheral IDUAe1 or IDUAe2 generated by transient hepatic expression led to elevated enzyme levels in capillary-depleted, enzyme-deficient brain tissues and protein delivery into nonendothelium perivascular cells, neurons, and astrocytes within 2 d of treatment. Moreover, 5 mo after long-term delivery of moderate levels of IDUAe1 derived from maturing red blood cells, 2% to 3% of normal brain IDUA activities were obtained in MPS I mice, and IDUAe1 protein was detected in neurons and astrocytes throughout the brain. The therapeutic potential was demonstrated by normalization of brain glycosaminoglycan and β-hexosaminidase in MPS I mice 5 mo after moderate yet sustained delivery of IDUAe1. These findings provide a noninvasive and BBB-targeted procedure for the delivery of large-molecule therapeutic agents to treat neurological lysosomal storage disorders and potentially other diseases that involve the brain.

Restricting transgene expression to maturing erythroid cells can reduce the risk for activating oncogenes in hematopoietic stem cells (HSCs) and their progeny, yet take advantage of their robust protein synthesis machinery for high-level protein production. This study sought to evaluate the feasibility and efficacy of reprogramming erythroid cells for production of a lysosomal enzyme, α-L-iduronidase (IDUA). An erythroid-specific hybrid promoter provided inducible IDUA expression and release during in vitro erythroid differentiation in murine erythroleukemia cells, resulting in phenotypical cross-correction in an enzyme-deficient lymphoblastoid cell line derived from patients with mucopolysaccharidosis type I (MPS I). Stable and higher than normal plasma IDUA levels were achieved in vivo in primary and secondary MPS I chimeras for at least 9 months after transplantation of HSCs transduced with the erythroid-specific IDUA-containing lentiviral vector (LV). Moreover, long-term metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in heart, liver, and spleen. Notably, neurological function and brain pathology were significantly improved in MPS I mice by erythroid-derived, higher than normal peripheral IDUA protein. These data demonstrate that late-stage erythroid cells, transduced with a tissue-specific LV, can deliver a lysosomal enzyme continuously at supraphysiological levels to the bloodstream and can correct the disease phenotype in both viscera and CNS of MPS I mice. This approach provides a paradigm for the utilization of RBC precursors as a depot for efficient and potentially safer systemic delivery of nonsecreted proteins by ex vivo HSC gene transfer.