IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy.

Pompe disease is caused by deficiency of acid α-glucosidase (GAA), resulting in glycogen accumulation in various tissues, including cardiac and skeletal muscles and the central nervous system (CNS). Enzyme replacement therapy (ERT) improves cardiac, motor, and respiratory functions but is limited by poor cellular uptake and its inability to cross the blood-brain barrier. Previously, we showed that hematopoietic stem cell (HSPC)-mediated lentiviral gene therapy (LVGT) with codon-optimized GAA (LV-GAAco) caused glycogen reduction in heart, skeletal muscles, and partially in the brain at high vector copy number (VCN). Here, we fused insulin-like growth factor 2 (IGF2) to a codon-optimized version of GAA (LV-IGF2.GAAco) to improve cellular uptake by the cation-independent mannose 6-phosphate/IGF2 (CI-M6P/IGF2) receptor. In contrast to LV-GAAco, LV-IGF2.GAAco was able to completely normalize glycogen levels, pathology, and impaired autophagy at a clinically relevant VCN of 3 in heart and skeletal muscles. LV-IGF2.GAAco was particularly effective in treating the CNS, as normalization of glycogen levels and neuroinflammation was achieved at a VCN between 0.5 and 3, doses at which LV-GAAco was largely ineffective. These results identify IGF2.GAA as a candidate transgene for future clinical development of HSPC-LVGT for Pompe disease.

Lentiviral gene therapy prevents anti-human acid α-glucosidase antibody formation in murine Pompe disease.

Enzyme replacement therapy (ERT) is the current standard treatment for Pompe disease, a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). ERT has shown to be lifesaving in patients with classic infantile Pompe disease. However, a major drawback is the development of neutralizing antibodies against ERT. Hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) provides a novel, potential lifelong therapy with a single intervention and may induce immune tolerance. Here, we investigated whether ERT can be safely applied as additional or alternative therapy following HSPC-LVGT in a murine model of Pompe disease. We found that lentiviral expression at subtherapeutic dose was sufficient to induce tolerance to the transgene product, as well as to subsequently administered ERT. Immune tolerance was established within 4-6 weeks after gene therapy. The mice tolerated ERT doses up to 100 mg/kg, allowing ERT to eliminate glycogen accumulation in cardiac and skeletal muscle and normalizing locomotor function. The presence of HSPC-derived cells expressing GAA in the thymus suggested the establishment of central immune tolerance. These findings demonstrate that lentiviral gene therapy in murine Pompe disease induced robust and long-term immune tolerance to GAA either expressed by a transgene or supplied as ERT.

Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning.

Fanconi anemia gene mutations are not involved in sporadic Wilms tumor.

Bi-allelic germline mutations of the Fanconi anemia (FA) genes, PALB2/FANCN and BRCA2/FANCD1, have been reported in a few Wilms tumor (WT) patients with an atypical FA phenotype. Therefore, we screened a random cohort of 47 Dutch WT cases for germline mutations in these two FA-genes by DNA sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Although several cases appeared to carry missense variants, no bi-allelic pathogenic mutations were identified, indicating that bi-allelic mutations in these FA-genes do not contribute significantly to the occurrence of WT.