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Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

Background: Hematopoietic stem cell transplantation (HSCT) recipients carry a high risk of primary varicella and varicella-zoster virus (VZV) reactivation. VZV infections can be fatal, and VZV reactivation can be complicated by postherpetic neuralgia, which worsens the recipients’ post-HSCT quality of life. VZV vaccines may prevent such infections; however, international vaccination guidelines do not recommend VZV vaccination of HSCT recipients because few data regarding safety among HSCT recipients are available. In Japan, we use a high-titer Oka stain vaccine for varicella vaccination and zoster prevention in the elderly; the plaque forming unit (PFU) value of this vaccine is as high as the zoster vaccine used in the U.S. Data regarding the safety of zoster vaccine in HSCT recipients is limited; therefore, we report our experience with this high-titer zoster-equivalent varicella vaccine in pediatric allogeneic HSCT recipients. Patients and Methods: Forty-seven pediatric allogeneic HSCT recipients who underwent transplantation at the Saitama Children’s Medical Center from 2000-2011 were vaccinated with live vaccines and their antibody titers were evaluated after vaccination. Among the 47 recipients, the Japanese high-titer varicella vaccine was administered to 32 recipients. Since establishing the live vaccine initiation criteria in 2009, allogeneic HSCT recipients were considered as recipients for live vaccines, including the varicella vaccine, if 24 months had passed after HSCT without active chronic GVHD or required immunosuppression. In addition, recipients were required to have a lymphocyte count >1500/μl or CD4 cell count >700/μl if they were 1000/μl or CD4 cell count >500/μl if they were >6 years, normal phytohemagglutinin response, and serum IgG level >500 mg/dL. Before 2009, time for vaccination depended on each doctor’s judgment. Patients were given a single dose of the Biken varicella vaccine containing a minimum of 23,000 PFUs. We use EIA to measure the antibody titer and defined seropositivity as a titer >6.0. The time to evaluate the antibody titer was not fixed. We retrospectively collected the clinical data of recipients and laboratory data by reviewing the medical records. Results: The 32 recipients, who received the Japanese varicella vaccine, underwent HSCT at a median age of 5.15 years (range: 0.5-16.4 years) and were vaccinated at a median of 20.65 months (range: 4.8-112.1 months) after HSCT. Twenty and 11 patients had received myeloablative and nonmyeloablative conditioning, respectively. Nine patients received related-donor transplant, of which eight were bone marrow and one was peripheral blood. Twenty-three patients received unrelated-donor transplant, of which 14 were bone marrow and nine were cord blood. Antithymocyte globulin was administered to four patients. Eighteen of the 32 patients (56.3%) were seropositive after vaccination. Of the 26 patients, whose vaccination histories and pre-transplantation histories were available, eight were affected by natural disease, six had been immunized, and 12 had not been immunized. At vaccination, the median lymphocyte count was 3201/μl (range: 817-6630/μl) and the median IgG value was 880 mg/dL (range: 233-1461 mg/dL). There was no significant risk factor associated with vaccine failure. We experienced one case of varicella because of wild type VZV and no cases of zoster after vaccination during a median follow-up period of 4.8 years (range: 0.4-11.3 years); the single patient developed varicella 13 days after vaccination, immediately after an influenza virus infection. Whereas, two cases of varicella and one case of zoster developed before vaccination. Of the other 15 recipients, who did not receive varicella vaccine, three varicella and two zoster developed in three recipients. Conclusion: We safely vaccinated pediatric allogeneic HSCT recipients with the Japanese high-titer varicella vaccine. This finding could encourage the use in the U.S. of zoster vaccines such as Zostavax@, which contains similarly high PFUs, and low-titer varicella vaccines such as Varivax@. However, the efficacy of high-titer varicella vaccines for preventing VZV reactivation in this population remains unclear. Further studies are warranted to elucidate the efficacy and difference between low-titer and high-titer varicella and zoster vaccines. Disclosures No relevant conflicts of interest to declare.

Abstract 4690 Hypophosphatasia (HPP) is one of the bone metabolic disorders caused by mutations of the liver/bone/kidney alkaline phosphatase (ALPL) gene encoding tissue-nonspecific alkaline phosphatase (ALP). The disease is characterized by the disturbance of bone and tooth mineralization and reduced serum ALP activity. Clinical severity of HPP often depends on the age of onset. Patients with perinatal and infantile forms usually have poor prognosis. Phenotype in patients with HPP is also closely related to residual enzyme activity affected by ALPL mutations. Most perinatal patients die of respiratory failure resulting from the reduced osteogenesis of the thorax, suggesting that production of new bones that support respiration in the chest would improve the prognosis. However, despite extensive studies on the molecular pathogenesis of HPP, there is no curative treatment for HPP. Mesenchymal stem cells (MSCs), which normally reside in bone marrow (BM), umbilical cord blood, muscle connective tissue, and adipose tissue, have multipotency that differentiate into various mesenchymal lineages and several mesoderm lineages. These include bone, cartilage, tendon, muscle, e adipose tissue, neuron, hepatocyte, and endothelium. MSC aloso modulates immune system and hematopoietic stem cell fate. These miscellaneous properties of MSCs are clinically applied such as prophylaxis for graft versus host disease (GVHD) or regeneration of ischemic heart because MSCs can easily be isolated and expanded in vitro while maintaining genetic stability. However, MSCs have never been used to facilitate bone formation or osteogenesis in patients with severe HPP. Herein we performed transplantation of MSCs expanded ex vivo for patients with severe HPP that underwent preceding BM transplantation (BMT) to determine the effects of MSCs on new bone generation. Two patients with severe hypophosphatasia were studied. Both patients were diagnosed as fatal HPP based on perinatal onset, respiratory disturbance, and ALPL mutations with extremely low ALP activity. MCSs obtained from these patients had little osteogenic activity in vitro. Their donors were asymptomatic relatives of the patients harboring heterozygous mutation of the ALPL gene. Osteogenic activity of donor-MSCs was normal. Myeloablative conditioning regimen was used for BMT. MSCs obtained from BM were expanded ex vivo in a flask containing a-minimum essential medium with 15% fetal bovine serum and 10 mg/mL kanamycin sulfate. The adherent cells became nearly confluent after 10 days and the expression of the cell surface antigens was mesenchymal type (CD34-, CD45-, CD105+, and SH3+). Case1: An 8-month-old girl was administered with buslfan (BU), fludarabine, and antithymocyte globulin (ATG). Tacrolimus, mycophenolate mofetil, and a short course of methotrexate (sMTX) were used for to GVHD prophylaxis. BMT was performed using marrow cells derived from a haploidentical donor. Cultured MSCs with cultured osteoblasts were administered 1 month after BMT. Although donor BM did not engraft, her respiratory condition was improved. Secondary MSCs transplantation (MSCT) was performed when her respiratory condition became deteriorated, which led to a significant improvement in her respiratory condition, her limbs length, and her physical development. Moreover, FISH and PCR analyses revealed that de novo formation of bone derived from both donor and recipient cells. Although, unexpectedly, she developed chronic myeloid leukemia with BCR-ABL fusions 2 years after BMT, she remained remission of the disease after secondary BMT from same donor. Mineralization of metaphysis improved although that of other bones such as long bones did not. Case2: A 14-month-old boy underwent BMT from a HLA 2 locus mismatched related donor using BU, cyclophosphamide, and ATG. Tacrolimus and sMTX were used for GVHD prophylaxis. Ex vivo expanded MSCs were transplanted 2 weeks after BMT. Engraftment of donor derived BM was confirmed. His condition gradually improved. Our data suggests that combination of MSCT and BMT may offer novel and effective treatment modalities to facilitate osteogenesis in patients with fatal HPP. Disclosures: No relevant conflicts of interest to declare.