Rescue of Lethal Hypophosphatasia Mice by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells.

Abstract 3154 Hypophosphatasia (HPP) is an inherited skeletal disease caused by genetic defects of tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme which is attached to the outside plasma membrane via a GPI anchor and plays an essential role in bone mineralization. The major symptoms are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Severe HPP is often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Although previous trials of ERT using various forms of soluble ALP showed no clinical benefit, it was recently demonstrated that TNALP with deca-aspartates at the C terminus (TNALP-D10) had a high affinity for bone tissue and repeated injections of TNALP-D10 successfully rescued lethal HPP mice. HPP mice were generated by knockout the mouse TNALP gene (Akp2) and phenotypically mimic to severe infantile HPP and develop hypomineralization, growth failure and epileptic seizures after birth. The plasma ALP activity in HPP mice was less than 0.01 U/ml (approx. 0.1 U/ml in wild type (wt) mice) and the average life span of non-treated HPP mice is about 20 days. We have also shown that a single intravenous injection of either lentiviral or AAV vector expressing TNALP-D10 resulted in prolonged survival and phenotypic correction of HPP mice. In this in vivo gene therapy, bone cells were not efficiently transduced, but the plasma ALP activity derived from TNALP-D10 secreted from transduced liver or muscle cells was maintained at extremely high levels (10 to 100 folds higher than that of wt mice). As an alternative approach, we are studying the feasibility of hematopoietic stem cells (HSC) based ex vivo gene therapy for HPP. After homing of HSC to the bone marrow, local expression of TNALP in the bone should be beneficial to improve bone mineralization. Other potential advantages of this strategy compared with an in vivo systemic gene therapy include lifelong expression of TNALP, no risk of germline gene transfer, and no immunoreaction against viral vector. Lineage negative bone marrow cells (BMC) were harvested from B6.CD45.1 mice (Ly5.1) using the Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD Bioscience) and incubated with lentiviral vector expressing GFP or TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6. Transduction efficiency assessed by GFP expression was approximately 40 % under the condition used. Recipient neonatal mice (Ly5.2) were sub-lethally irradiated at 4Gy and received BMC (1 × 106̂ cells) through the jugular vein on day 2. Irradiated neonatal wt mice showed a slight reduction of the growth rate but normal physical activity and healthy appearance. GFP positive donor cells migrated to the bone marrow in recipient mice. FACS analysis of the peripheral blood samples 4 to 12 weeks after transplantation demonstrated that approximately 30 % of Ly5.1 donor cells were stably detected in all lineage blood cells of recipient mice. After treatment of neonatal HPP mice with TNALP-D10 expressing BMC, the plasma ALP activity was elevated to 1 to 2 U/ml at 4 weeks of age and remained at this level during the observation period. The treated mice actively moved in the cage without epileptic seizures and the life span was prolonged over 3 months. X-ray examination of the skeleton showed that mineralization was significantly improved compared to non-treated HPP mice, but not completely normalized compared to age matched wt mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for continuous supply of TNALP-D10 to rescue lethal HPP mice. However, the concentration of TNALP-D10 in the bone may not be sufficient for complete correction of skeletal abnormalities. Further optimization of gene transfer and neonatal BMT is under way to increase the plasma ALP activity. HSC mediated ex vivo gene therapy is now being applied to treat not only hematological diseases but also neurological disorders such as adreno leukodystrophy and metachromatic leukodystrophy. Hypophosphatasia, a systemic bone disease, is also an important target for ex vivo gene therapy. Disclosures: No relevant conflicts of interest to declare.