Bone Marrow Cell Based Enzyme Replacement Prolongs Survival and Improves Disease Phenotypes In a Mouse Model Of Lethal Hypophosphatasia

Hypophosphatasia (HPP) is an inherited skeletal disease caused by mutations of the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme and plays an essential role in bone mineralization. The major symptoms of HPP are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Perinatal and infantile forms of HPP are often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Currently, Phase II/III clinical trials of ERT using a recombinant TNALP which linked deca-aspartate (D10) at the C terminus for bone targeting are ongoing in North America, Europe and Japan. The perinatal and infantile patients received the ERT showed apparent improvement of the symptoms. However, the ERT is highly invasive for the young patients because it requires repeated subcutaneous administration of large amounts of the enzyme every 3 times a week for long-term correction. As another approach to treat HPP, we have reported in vivo gene therapy for ALPL ( Akp2 ) knock-out mice (HPP mice). The treated HPP mice were rescued by a single systemic injection of lentiviral vector or adeno-associated viral vector expressing bone targeted form of TNALP (TNALP-D10) during the neonatal or fetal period. Although untreated HPP mice developed apparent growth failure and died by around 20 days of age due to severe skeletal hypomineralization and epileptic seizure, the treated HPP mice were prolonged the survival and improved the physical activity. In the treated HPP mice, plasma ALP activity was kept higher than 1 U/ml (approximately 0.01 U/ml in untreated HPP mice and 0.1 U/ml in wild type (WT) mice) which gives therapeutic effects. However, disadvantages of in vivo gene therapy include the risk of germline gene transfer and induction of immune responses to the vectors or transgene products. To overcome these problems, we examined a feasibility of ex vivo gene therapy using hematopoietic stem cells (HSC) transduced by lentiviral vector expressing TNALP-D10. The potential advantages of this approach are lifelong expression of TNALP-D10 and prevention of risks of in vivo gene therapy. The lineage negative bone marrow cells containing HSC (Lin- BMC) were harvested from B6.CD45.1 mice (Ly5.1) and then enriched using Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD bioscience). Lin- BMC was transduced with lentiviral vector expressing TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6 on Retronectin coated plate. Recipient HPP mice (Ly5.2) on day 2 after birth were received a sub-lethal dose of total body irradiation (4Gy) 4hr prior to transplantation. Then, the transduced Lin- BMC (1 x 106 cells) was transplanted intravenously into the HPP mice through the temporal vein or jugular vein. The plasma ALP activity was rapidly elevated approximately 400 fold higher than untreated HPP mice (untreated: 0.014±0.004 units/ml (n=4) and treated: 5.39±2.29 units/ml (n=7), respectively) on 1 week after the transplantation and kept at this level during the observation period. Engraftment rate of Ly5.1 donor cells were sustained at approximately 30-40% with multilineage potential. The treated HPP mice were prolonged their survival over 3 months without epileptic seizures and the physical activities were improved. The histochemical ALP staining indicated TNALP-D10 was accumulated on the surface of trabecular and cortical bones of the treated HPP mice. The bone mineralization was significantly improved, but still not satisfactory compared with age matched WT mice. Contrary to our expectations, 2 of 9 HPP mice transplanted with non-transduced BMC also survived for 3 months. However, the plasma ALP activity was not elevated at all and the bone mineralization was incomplete compared with treated HPP mice. These results indicate that a single transplantation of genetically modified BMC at neonatal period is sufficient for long-term supply of TNALP-D10 and rescue of lethal HPP mice, even though hypomineralization was not completely recovered. Further optimization of viral vector and conditioning of transplantation is required to increase the treatment efficacy for HPP. However, neonatal ex vivo gene therapy using genetically modified BMC would be a possible and practical approach to treat HPP. Disclosures: Watanabe: Alexion Pharmaceuticals, Inc.: Membership on an entity’s Board of Directors or advisory committees.