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Adrenoleukodystrophy (ALD) is an X-linked peroxisomal disorder of very long chain fatty acid accumulation. The cerebral form of ALD is associated with inflammatory brain demyelination producing visual, auditory and cognitive processing defects along with loss of motor function. Death occurs within two to five years of clinical onset, unless the patient receives hematopoietic cell transplantation (HCT).As bothALDandHCTare associated with oxidative stress, we reasoned that adjunctive therapy with an antioxidant agent, N-acetyl-L-cysteine (NAC; used for several decades as mucolytic agent in bronchopulmonary illnesses and as an antidote for liver injury from acute acetaminophen toxicity), may provide protection from neurological decline observed prior to stabilization after successful HCT. Our previous findings suggested that peri-transplant administration of NAC improves survival in advanced cerebral ALD (as defined by MRI severity score, MRISS, e 10). Thus, we investigated the safety of NAC administration in boys with early cerebral ALD (MRISS 100 days after HCT. The implication of our findings is that administration of NAC is safe in childhood ALD recipients of HCT. NACmerits investigation as a therapeutic strategy to reduce oxidative stress within the central nervous system in HCT recipients with lysosomal storage disorders affecting the central nervous system.

MPS IH (Hurler syndrome) is caused by severe mutations in a-L-iduronidase gene, leading to accumulation of complex sugars, termed glycosaminoglycans (GAG), in tissues. The disorder is lethal unless treated with hematopoietic stem cell transplantation (HSCT). When compared to other recipients of HSCT, MPS I patients are well-known to have an increased incidence of bleeding complications in peri-transplant period. To investigate whether this bleeding propensity is related to the enzyme deficiency and GAG accumulation we evaluated five MPS IH patients (2 females and 3 males, mean age: 1.1 years) prior to any therapy. We observed that three of the five patients had an elevated activated partial thromboplastin time (aPTT, normal range 22–35 seconds). As no heparin was used in any of these patients, we reasoned that the accumulation of heparan sulfate, a hallmark of MPS IH, may serve as a heparin-like anticoagulant. Should this assumption be correct, higher levels of GAG would be expected in the three patients with the elevated aPTT. This, in fact, was what we have observed (Figure), suggesting that dose - effect relationship exists between whole body GAG load (assessed as milligrams of urinary GAG normalized to grams of creatinine; x-axis) and level of anticoagulation (aPTT in seconds; y-axis). Figure Figure In this context, it is important to note that the patient with the second highest value of urinary GAG and aPTT experienced pulmonary bleeding on day 12 after HSCT requiring intubation and ventilatory support for 25 days. We hypothesized that endogenous GAG heparan sulfate exerts its antithrombotic effect through antithrombin III-mediated inhibition of factor Xa, as in clinically used low molecular weight heparinoid, danaparoid. To test this hypothesis, we have assessed anti-Xa levels in mice with a-L-iduronidase deficiency, an animal model of MPS IH. As expected, the anti-Xa level were not significantly different in wild type C57Bl/6 mice (N=3) and heterozygous mice (N=6): 0.11±0.005 IU/cc plasma versus 0.10±0.004 IU/cc plasma (p=0.2). Anti-Xa values of MPSI mutant mice (N=14), however, were significantly elevated when compared to the age-matched sex-matched wild type controls (0.19±0.05 IU/cc plasma versus 0.11±0.005 IU/cc plasma; p

In addition to the well established role of hematopoietic stem cell transplantation (HSCT) in the treatment of malignancies, HSCT is utilized for a number of neurologic metabolic diseases (typically, enzyme deficiencies) due to the ability of donor graft to provide an ongoing source of enzyme that can be taken up by recipient’s cells. Many of these diseases damage the white matter of the brain, and after onset of symptoms are characteristically progressive and lethal. Major limitations to the success of HSCT as therapy for these diseases are graft failure and toxicity to the brain from conditioning, resulting in disease progression. Thus, we reasoned that it would be advantageous to develop a less intensive conditioning regimen that minimally contributed to central nervous system toxicity while providing sufficient immunosuppressive to permit engraftment from unrelated cord blood donors. We are testing a reduced intensity regimen including Campath 1H (1.5 mg/kg), clofarabine (200 mg/m2), melphalan (140 mg/m2), and low dose total body irradiation (200 cGy) for this patient population. To gain insight into the persistence of Campath 1H, we measured serum levels within 30 hours prior to the cord blood infusion. We report outcomes in three adults and three children: | | Diagnosis | Age (years) | UCB Graft; HLA match | NC dose (×108/kg | CD34+ dose (×106/kg) | Campath 1H serum level* | Donor Engraftment | |:- | --------- | ----------- | -------------------- | ---------------- | -------------------- | ----------------------- | ----------------- | | 1 | MLD | 44 | dUCB; 4/6, 5/6 | 0.44 | 0.68 | Undectectable | 0% | | 2 | ALD | 9 | dUCB; 5/6, 5/6 | 0.90 | 1.43 | 699 | 100% | | 3 | ML | 1 | dUCB; 5/6, 5/6 | 1.43 | 3.04 | 1,575 | 100% | | 4 | TS | 1 | sUCB; 6/6 | 0.97 | 1.33 | 2,250 | 100% | | 5 | MLD | 42 | dUCB; 5/6, 6/6 | 0.76 | 0.94 | 2,728 | 60% | | 6 | MLD | 42 | dUCB; 5/6, 5/6 | 0.22 | 0.24 | Not done | 100% | Legend: MLD, metachromatic leukodystrophy; ALD, adrenoleukodystrophy; ML, mucolipidosis type II; TS, Tay-Sachs disease; NC, nucleated cell; dUCB, double unit umbilical cord blood; sUCB, single unit umbilical cord blood; ND, not detected; Eng, engraftment; HLA matching is reported for antigen level HLA-A, B and allele level DRB1. *Campath 1H levels (nanograms/mL) are reported as an average of two measurements of samples diluted 10x. Cumulative doses for both UCB units are shown. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed grade II skin and gastrointestinal acute GvHD, treated successfully with systemic and topical steroids. All patients are alive; none experienced progressive deterioration of neurologic function in the peri-transplant period. Our data suggest that this conditioning regimen is minimally toxic to the brain. The two subjects with the lowest Campath 1H concentrations had autologous recovery (patient 1) and GvHD (patient 2), suggesting that in vivo T cell depletion with Campath 1H may be beneficial. These results suggest that novel modifications in the transplant process may provide opportunities to decrease neurologic toxicity and maintain optimal neurologic function in this high risk population.

Dystrophic epidermolysis bullosa (EB) is a disorder of incurable skin fragility, blistering and perturbations in anchoring fibrils caused by mutations in the type VII collagen gene (COL7A1). Since bone marrow (BM) includes cells with capacity to differentiate into diverse cell types and can seed various organs, including skin, we hypothesized that if type VII collagen producing cells are present in BM, BM infusions could be a beneficial therapy for EB. To test this hypothesis this in vivo, we assessed multiple candidate populations for cellular therapy of murine recessive dystrophic EB (RDEB, col7a1 −/−). As the col7a1 −/− newborn mice develop blisters and do not survive after two weeks of life, we can readily identify the mutant pups and the lethality provided a robust readout for the cellular interventions. The following cell populations were tested: epidermal stem cells and transit amplifying cells (from epidermis); total BM; BM derived multipotent adult progenitor cells and mesenchymal stem cells; fetal liver cells; and lineage positive and lineage negative BM fractions. None of these cell populations, however, provided any correction in col7a1 −/− recipients (N=223), even when the infusion regimen was varied by cell dose, age of donor, in utero and postnatal age at the time of infusion, and intravenous versus intrahepatic infusion. In a search for alternatives, we used CD150+ and CD48− BM cells, since signaling molecule SLAM family receptor positive populations have been shown to have significant pluripotentiality. We used several doses and infused them early postnatally into unconditioned col7a1 −/− mutant animals. One of these conditions, namely CD150+/CD48− BM population from C57Bl/6 GFP transgenic mice at a dose of 8 million cells per animal administered on day 3 or 4 after birth intravenously, resulted in survival of 3 out of 13 (23%) animals in two independent experiments. One of them was harvested at postnatal day 55; two of them were electively harvested at postnatal day 70. The animals were confirmed to be mutants by genotyping using PCR. The weights of these three animals were lower than that of their normal litter mates. Donor engraftment was up to 3% in peripheral blood and up to 12% in BM. Strikingly, the skin blisters characteristic of EB healed. Tissue analyses showed that the adoptive transfer of donor SLAM CD150+ selected BM resulted in skin engraftment of GFP+ donor cells and in the production of VII collagen mRNA in the skin, as assessed by RT- PCR. BM cells explanted from the 3 surviving recipients showed collagen VII producing cells. Ultrastructurally, we were able to identify anchoring fibrils, structures in the skin that are comprised exclusively of type VII collagen protein. This is consistent with the hypothesis that adoptive transfer of enriched wild-type BM cells resulted in, at least partial, functional correction of RDEB. Experiments are ongoing to identify the type VII collagen producing cells in CD150+ and CD48− BM population, and, in parallel, to identify immature BM cells that could be induced into becoming a skin stem cell. Collectively, these data demonstrate proof-of-principle of BM transfer for correction of the basement membrane zone defect in col7a1 −/− animals that may offer a valuable approach for treatment of human RDEB.

Dyskeratosis congenita (DC) is a congenital disorder characterized by very short telomeres. Clinical presentation includes a diagnostic triad (lacey reticular pigmentation, nail dystrophy, and leukoplakia), aplastic anemia (the main cause of premature death), myelodysplastic syndrome/leukemia and solid tumors. Allogeneic HCT is the only curative option for the hematologic complications of DC but has been associated with a high risk of peri-transplant morbidity and early death. Only about fifty HCT for DC have been performed to-date, and the five year survival after related donor HCT has been about 75%, but only approximately 35% when an unrelated donor was used. To improve survival in DC patients by decreasing transplant mortality, we introduced a reduced intensity regimen including cyclophosphamide (50 mg/kg), fludarabine (200 mg/kg), low dose total body irradiation (200 cGy), and (in patients 3 and 4) campath 1H (1 mg/kg). To decrease the risk of graft rejection, grafts were not T-cell depleted. We report outcomes in four consecutive patients, two adults and two children, all of whom engrafted with donor hematopoiesis: Age (years) Sex Graft and HLA match NC dose (×108/kg) CD34 dose (×108/kg) Follow-up (months) Donor chimerism 24 M URD dUCB 4/6, 4/6 0.55, 0.39 0.5, 0.43 1 (dead*) 83% 29 F REL PBSC 6/6 13.93 4.43 20 (alive) 100% 5 F URD BM 7/8 1.38 1.55 16 (alive) 100% 2 M URD BM 8/8 5.92 2.31 3 (alive) 100% Legend: M, male; F, female; NC, nucleated cell; URD, unrelated donor; REL, related donor; BM, bone marrow; dUCB, double umbilical cord blood; PBSC, peripheral blood stem cells; *Patient had autologous recovery after the first dUCB and died of sepsis 1 month after the second dUCB; HLA matching is reported for antigen level HLA-A, B and allele level DRB1 for cord blood, and allele level typing for HLA-A, B, C, DRB1 for PBSC or BM. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed limited chronic GvHD and patient 4 developed grade III skin acute GvHD. Both were treated successfully with systemic and topical steroids. Our data suggest that this conditioning regimen results in a low rate of transplant related complications without compromising engraftment. Critically, early fatal pulmonary and vascular complications, common in post-transplant courses in DC patients, were not observed. This highlights the need to avoid drugs that are associated with pulmonary toxicity such as busulfan, and to limit radiation to the lung in patients with DC. This new less intensive conditioning regimen appears to result in a low rate of transplant related complications, and yet has adequate immunosuppressive activity to permit engraftment from alternative donors in DC patients.

Determination of the degree of somatic mosaicism providing functional correction of Fanconi anemia (FA) hematopoiesis has direct implications for gene therapy for FA: it may help assess the percentage of FA hematopoietic cells corrected by gene therapy approaches that are needed to achieve clinically meaningful effects. Hypersensitivity to DNA interstrand cross-linking agents, such as diepoxybutane (DEB) and mitomycin C (MMC), is a cellular marker for diagnosis of FA. However, in some FA patients a population of DEB-resistant PHA-stimulated lymphoblasts (PHA-L) was observed, and this population sometimes varied over time. To assess the significance of this finding on hematopoietic function, we evaluated the MMC sensitivity of bone marrow mononuclear cells (BMMC) and DEB sensitivity of PHA-L and cultured lymphoblastoid cell lines (LCL) in 42 consecutive FA patients referred to the University of Minnesota. In cases where LCL were DEB-resistant, cultured fibroblasts were also studied. BMMC were cultured in the presence of increasing concentrations of MMC. PHA-L and LCL were cultured in DEB at 0.1 mcg/ml. Wild type BM progenitors (N = 17 subjects) proliferated regardless of increasing MMC concentrations (albeit at decreased efficiency at the highest concentrations) as follows: 0 MMC (normalized to 100%), 5 nM MMC (99% [standard deviation, SD, 16%]), 10 nM MMC (90% [SD 22%]), 25 nM MMC (77% [SD21%]), and 50 nM MMC (44% [SD 30%]). Of the 42 FA patients, BMMC failed to proliferate at 0 nM MMC in 10 patients and at 5 nM MMC in 20 patients. Twelve FA patients had MMC resistant BMMC: cells cultured in 5, 10, 25 and 50 nM MMC grew 44% (SD 28%), 35% (SD 24%), 24% (SD 30%) and 17% (SD 32%) of colony numbers in MMC free culture, respectively. Six of these 12 subjects were PHA-L mosaics as determined by DEB sensitivity testing. Four patients with no growth of BMMC at 0 or 5 nM MMC were also somatic mosaics in their PHA-L and LCL. Thus there was no clear correlation between somatic mosaicism as demonstrated by DEB testing in peripheral blood and sensitivity of BMMC to growth in MMC. Clinically, two patients with hematopoietic somatic mosaicism developed severe marrow aplasia, one of which received hematopoietic stem cell transplantation. Four of the mosaic patients had normal or near normal peripheral blood counts with one patient having clonal hematopoiesis by HUMARA assay and only low levels of metaphases with multiple breaks in multiple DEB studies. While patients with hematopoietic somatic mosaicism had mixed populations of DEB sensitive cells in their peripheral blood, all their fibroblast cultures were DEB sensitive. In summary, these data show that the presence of somatic mosaicism per se does not necessarily prevent bone marrow failure. Moreover, the data suggest that patients with stigmata of FA may have chromosomal breakage studies showing few cells (or no cells) with the characteristic changes of FA; in these cases, skin fibroblasts should be tested as well.

Acute lymphocytic leukemia (ALL) is the most common childhood leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improved cure rates in both pediatric and adult ALL patients. In this study, we show human B-lineage acute lymphocytic leukemia (B-ALL) cell lines (6/6 tested) and CD19+CD10+ primary B-ALL cells from patients (8/8 tested) express TLR7 mRNA by real-time RT-PCR and TLR7 proteins by Western blot. Triggering TLR7 on B-ALL cells with a TLR7 agonist (imiquimod) significantly increases the cell surface expression of molecules essential for T cell activation (CD40, CD54, CD80, CD86, and HLA-DR), the ligands for NKG2D and ligands for natural cytotoxicity receptors (NKp30, NKp44, and NKp46) which regulate NK-mediate killing. Thus, TLR7 signaling enhances the immunogenicity of B-ALL cells and makes them more suitable targets for T cell and NK cell mediated attack. Most importantly, TLR7 agonists strongly suppress in vitro growth of B-ALL cell lines (RS4;11, BLIN-1) and induces profound apoptosis of primary B-ALL cells from patients in culture in a TLR7 agonist dose-dependent manner. Both t(4;11)-positive RS4;11 cells and t(4;11)-negative BLIN-1 cells proliferate rapidly in culture with a 30–40 fold increases of leukemia cell number in 7 days. The addition of TLR7 agonist at 10 ug/ml fully inhibit the growth of RS4;11 and BLIN-1 cells in culture. Furthermore, TLR7 agonist treatment dramatically induces apoptosis of primary B-ALL cells isolated from the patients (2/2 with t(9;22), 6/6 without t(9;22)) with 0.4%–13.3% leukemia cells left at day 5 of culture. The TLR7 agonist-mediated apoptotic death of B-ALL cells was conformed by viable cell counts, TMRE staining, and, Western blots of the activation and cleavage of caspases. To study the in vivo therapeutic effects of TLR7 agonist against human B-ALL, RS4;11 and BLIN-1 cells were luciferase labeled and injected into NOD/SCID mice. Both RS4;11 and BLIN-1 leukemia cells engrafted in multiple organs (BM, spleen, liver, lymph nodes, kidney) resulting in uniform lethality of RS4;11 mice in 8 weeks and BLIN-1 mice in 12 weeks, respectively. Flow cytometry and tissue staining results confirmed that these organs were massively infiltrated with human CD45+19+ leukemia cells. To determine whether TLR7 preincubation of RS4;11 or BLIN-1 cells would prolong survival due to an apoptotic effect, cohorts of mice were injected with a lethal dose of RS4;11 or BLIN-1 cells with or without pre-incubation with TLR7 agonist. Mice receiving TLR7 agonist pre-pretreated B-ALL cells had a significant increase in long-term survival rate and significant reduction in tumor burden at the time points evaluated. These in vivo results confirm previous in vitro findings and suggest that TLR agonist-treated B-ALL cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established B-ALL is being investigated using these xenograft mouse models. These results form the basis for a clinical trial of systemic TLR7 agonist administration for treating patients with B-ALL. In summary, we have shown that TLR7 targeting increases B-ALL immunogenicity and directly induces B-ALL apoptosis, providing new insights into the biology and therapy of B-ALL.

The beneficial effects of Mesenchymal Stem Cells (MSC) are being tested clinically in attempts to improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and to deliver therapy for malignancies. In early reports, phase I clinical studies have not been associated with toxicities. To study the biodistribution of MSC, we labeled adult murine C57BL/6 MSC with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused labeled MSC with bone marrow into irradiated allogeneic recipients. Using in vivo whole body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in lungs. Two mice also developed sarcomas in their extremities. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Mapping of the Sleeping Beauty transposition insertion sites did not identify an obvious transposon-related genetic abnormality. Importantly, the original MSC cultures not labeled with transposons, as well as cultured MSC independently isolated from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro. Even though not all MSC cultures formed tumors upon in vivo injection, these data indicate that MSC transformation was neither strain-specific nor a rare event following ex-vivo expansion. Karyotype analyses using fluorescence in situ hybridization with spectral karyotyping (SKY) as well as combined binary ratio labeling of nucleic acid probes (COBRA) showed clonal evolution of transformed MSC suggesting that the critical transformation event(s) occurred before MSC infusion. Collectively, we describe cytogenetic instability of murine MSC isolated in two independent laboratories, their cellular transformation, and potential for sarcoma formation. While the growth characteristics of human and murine MSC are not identical and murine cells are more prone to undergo immortalization and transformation in culture than human cells, our study highlights the importance of quality control measures needed for ongoing and future clinical trials using human MSC.

9040 Background: Post-transplant lymphoproliferative disorder (PTLD) is a known complication of immunosuppression following solid organ and stem cell transplantation. It ranges from benign lymphoid hyperplasia to fulminant systemic disease with high mortality. Most cases are B lineage and associated with Epstein-Barr virus (EBV+). T cell PTLD is rare and usually EBV negative. To date only 5 cases of EBV+ T cell PTLD have been reported in pediatric patients and none have had documented involvement of the central nervous system (CNS). Methods: We provide clinical, histologic, immunophenotypic and molecular details of a case of fulminant EBV+ T cell PTLD with CNS involvement and compare our data to the clinical presentations, biology and outcomes of the other reported cases of pediatric T cell PTLD (these include 5 EBV+ CNS− cases, 3 EBV− CNS+ cases, and 4 EBV− CNS− cases with dissemination or marrow involvement). Results: A 4.5 year old male developed EBV+ T cell PTLD with CNS involvement 3 years following a cadaveric renal transplantation. His fulminant presentation included fever, hypotension, splenomegaly, pancytopenia, coagulopathy, and bilateral pleural effusions. He had lymphocytosis in his CSF and his MRI showed brain white matter changes consistent with leukoencephalopathy. T lineage was confirmed by the presence of T cell markers CD3, 5, 7, 45 and TCRγ. The presence of EBV was demonstrated by in situ hybridization for EBER. His treatment followed the Children’s Oncology Group protocol A5971 for disseminated lymphoblastic lymphoma that employs a standard NHL/BFM-95 regimen with cyclophosphamide and anthracycline intensification during the induction and delayed intensification phases with a treatment duration of 2 years. He has received no irradiation. After 16 months, he has no measurable disease. Conclusions: EBV+ T cell PTLD is extremely rare in pediatrics and has resulted in mortality in 12 of 17 reported cases. This is the first report of EBV+ T cell PTLD with CNS involvement in a pediatric patient. Although no standardized treatment exists, the fulminant presentation, T lineage disease, and CNS involvement warranted aggressive systemic and intrathecal chemotherapy. No significant financial relationships to disclose.

KIR-ligands are HLA molecules that can be grouped into 3 major categories based on the amino acid sequence determining the KIR-binding epitope in HLA-C and HLA-B alleles. Almost all HLA-C alleles are of the C1 or C2 group defined by single nucleotide polymorphisms (SNPs) found at amino acid positions 77 and 80. C1 is designated S77 and N80, while C2 is designated N77 and K80. Most HLA-B alleles can be classified as either Bw4 or Bw6, defined by SNPs located at amino acid position 77 and 80–83. Bw6 allele types which are invariant and consistently S77 and N80 are not KIR-ligands, in contrast to Bw4 which is a KIR-ligand and can have multiple amino acid sequences at these positions. Killer-immunoglobin receptors KIR2DL1, KIR2DL2 and KIR3DL1 bind KIR-ligands C1, C2 and Bw4 respectively, resulting in inhibition of NK cell mediated lysis. Recent transplant strategies based on KIR-ligand mismatch to predict NK cell alloreactivity have resulted in less relapse and better survival in patients with AML. Although allele level high-resolution HLA-typing is the gold standard for accurately determining KIR-ligand status, it is not performed at some centers and is cost prohibitive for retrospective cohorts. In these settings, KIR-ligand assignment is being extrapolated from serologic or low-resolution HLA data. This is only accurate in about 80% of donor/recipient pairs, because misclassifications based on the frequency of less common alleles can result in assignment to the opposite KIR-ligand group (Bw4 versus Bw6 or C1 versus C2). Our aim was to develop a high-throughput assay for determining KIR-ligand status which is accurate, inexpensive and rapid. Pyrosequencing is a relatively new method for sequencing DNA and is especially useful in detecting SNPs when most of the sequence is already known. Sequencing is based on a single strand of biotinylated DNA which is used as a template for a sequencing primer specific to the region of interest. As nucleotides are added base by base from the sequencing primer, the pyrosequencing apparatus (PSQ MA 96) can detect which base is added and in what quantity. This information can be used to determine if the sample is homozygous or heterozygous. We hypothesized that pyrosequencing would be a viable alternative to high resolution HLA-typing for KIR-ligand status determination. It directly sequences the ligand epitopes, thus avoiding misclassifications encountered with low resolution HLA-typing alone. This high throughput system would be of particular interest in analysis of banked RNA and DNA tissue samples for retrospective cohorts when high resolution typing is not available. KIR-ligand status was assigned for 34 samples by testing with both pyrosequencing and high-resolution HLA-typing. Initially we found a discrepancy rate of 9% between the two methods. To investigate discrepant samples, PCR products from these reactions were sequenced. We concluded that the initial PCR primer set was designed over a polymorphic region which did not amplify all known HLA-B or HLA-C alleles. The PCR primers were redesigned and the samples retested by pyrosequencing, resulting in full concordance with high resolution HLA data. Pyrosequencing is a sensitive, specific, high-throughput and inexpensive screening technique to rapidly determine KIR-ligand status for evaluating potential alloreactive NK cell or transplant donors.

The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

MAPCs are pluripotent cells derived from mesenchymal stromal cells (MSCs) in adult bone marrow. In contrast to MSCs, MAPCs differentiate into various lineages of mesodermal, ectodermal and endodermal origin, and contribute to numerous terminally differentiated tissues in the recipients. This capacity is enhanced in the setting of injury, suggesting a possible role of MAPCs in repair and regeneration in disease states. We aimed to investigate the capacity of MAPCs to aid in myocardial repair in hearts with postinfarction remodeling. We reasoned that as MAPCs differentiate to both endothelium and cardiomyocytes in vitro and as engraftment of delivered cells depends on establishing adequate blood flow in the ischemic region, MAPCs may represent the optimal cell type to contribute to both angiogenesis and the parenchymal tissue to regenerate function of injured myocardium. To study engraftment and survival of MAPCs, we labeled adult murine C57BL/6 MAPCs with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and injected them into myocardium of C57BL/6 adult mice with acute myocardial infarction (AMI). Mice were anesthetized, intubated and mechanically ventilated using a small-animal respirator. Under a stereomicroscope the heart was accessed via left thoracotomy. The left anterior descending coronary artery was ligated at mid-level between apex and base with a 9-0 surgical suture to produce AMI. Twenty minutes later, intramyocardial injections of labeled MAPCs in saline, or saline alone were administered at five distinct injection sites at boarder zone of AMI (total MAPC dose = 106/mouse). Chest was closed in layers and animals were allowed to recover. Mice were followed with echocardiography and in vivo whole body bioluminescent imaging. Seventy days after AMI, MAPCs recipients (N=6) had significantly less severe left ventricular (LV) dilatation evidenced by a smaller LV end-diastolic and LV end-systolic dimensions when compared to control mice infused with saline (N=4) (average±standard deviation, 4.7±0.2 mm versus 5.3±0.5 mm, p=0.05; and 3.7±0.2 mm versus 4.4±0.6 mm, p=0.03, respectively). In addition, ejection and shortening fractions were significantly higher in MAPC recipients (36±2% versus 30±3%, p=0.004; and 20±1% versus 16±2%, p=0.004, respectively). Luciferase signals emitted from donor MAPCs were easily detectable in MAPC recipients 100 days after MAPC infusion, at which point the animals were harvested. Analyses are ongoing to determine whether MAPCs and their progeny contributed to expansion of coronary vasculature (capillary density), or formed or modified injured myocardial tissue. Alternatively, both populations of repair cells could have been derived from donor (DsRed2+) MAPCs, or donor MAPCs could have provided permissive local environment to recruit recipient cells and enhance endogeneous regeneration. In summary, these findings provide evidence that MAPCs persist long term in injured myocardium and document the potential of MAPCs for improvement of cardiac function after ischemic myocardial injury. Jakub Tolar and Xiaohong Wang contributed equally to this study.

Epstein Barr Virus (EBV) associated post-transplantation lymphoproliferative disease (PTLD) is commonly treated with reduction of immune suppression, Rituximab and chemotherapy, but few options exist for individuals with resistant disease. Donor lymphocyte infusions (DLI) from the patient’s original donor have provided responses in PTLD following allogeneic marrow transplantation, but HLA matched allogeneic donors are generally not available for solid organ transplantation recipients. A renal-pancreas transplant recipient with refractory PTLD was treated with an infusion of haploidentical T cells genetically engineered to allow eradication of these cells by the administration of ganciclovir should severe GVHD or organ rejection be observed. A retrovirus capable of expressing the herpes simplex virus thymidine kinase (HSV-tk) gene to provide negative selection and the human nerve growth factor receptor (NGFR) for positive selection was used to transduce donor T cells (1.2 x 109) 48 hours post activation with anti-CD3/CD28 beads. The transduction frequency was 37.2% based on NGFR expression; following selection with a biotinylated anti-NGFR antibody and anti-biotin magnetic beads using the Miltenyi CliniMACS a 98.1% NGFR+ product was obtained. Engineered T cells (106/kg) were infused following a lympho-depletion regimen consisting of fludarabine 125 mg/m2 and cyclophosphamide 60 mg/kg. Platelet (>50,000) and neutrophil (>500) recovery occurred 10 and 12 days post cell infusion, respectively. A bone marrow evaluation on day +21 revealed 8.2% of nucleated cells in the marrow to be of donor origin by VNTR analysis. On day +30 peripheral blood VNTR testing documented 28.6% of mononucleated cells to be donor in origin. CT imaging 4 weeks after infusion of the engineered T cells documented decreased size of the primary tumor in the breast. After the initial neutrophil and platelet recovery, neutropenia and thrombocytopenia recurred. A bone marrow evaluation on day 37 revealed decreasing cellularity, possibly consistent with GVHD associated aplasia; VNTR analysis of this marrow sample revealed 20.9% of cells to be of donor origin. The decision was made to treat the patient with ganciclovir (GCV) to eliminate donor T cells contributing to aplasia. Unfortunately, the patient expired within 48 hours of beginning GCV due to an infectious complication. On autopsy, no evidence of previously biopsy proven EBV PTLD was found in the lung and the buccal surface of the mouth. In addition, much of the primary breast mass was free of PTLD. Lymphocytic infiltration of hepatic portal tracts was observed, consistent with GVHD. No evidence of rejection was noted in the transplanted kidney or pancreas. In summary, an excellent response of a refractory EBV PTLD was obtained using therapy including haploidentical T cells engineered for negative selection, and extended persistence of the cells was documented was for greater than 1 month. In the future, a secondary drop in blood counts will be used to start CGV for negative selection of these cells in vivo. This novel strategy may provide effective therapy to transplant recipients without donors who invariable die of their underlying secondary malignancy.

Mesenchymal stem cells (MSCs) can differentiate into non-hematopoietic cell types, including adipocytes, chondrocytes and osteocytes. MSCs have been isolated from multiple species, including humans, and multiple organs, including bone marrow, adipose tissue and umbilical cord blood. The beneficial effects of MSCs are being tested clinically in attempts to: improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and as antitumor agents to deliver therapy for malignancies. Phase I clinical studies have not been associated with toxicities. We aimed to investigate the capacity of MSCs to aid in tissue healing after radiation induced injury in irradiated bone marrow transplant (BMT) recipients. To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused them with allogeneic bone marrow into irradiated reipients. Using in vivo whole body bioluminenscent imaging luciferase signals were shown to be increased between weeks 3 and 12 indicating expansion of MSCs. Unexpectedly, some mice (N=8/17) with the highest luciferase signals died and all surviving mice (N=9/17) developed foci of ectopic ossification in lungs. Two of mice also developed osteosarcomas in their extremities. This prompted us to characterize the transformed MSCs that originated from the donor MSCs. The transformed cells were aneuploid, lost their capacity to differentiate into mesenchyme-derived adipocytes and chondrocytes, and histologically identified as osteosarcomas. In addition, infusion of tumor cells resulted in malignant lesions in secondary recipients. Mapping of transposition sites in the genome and karyotype analysis indicated that the critical transformation event(s) occurred before infusion of the MSCs. Even though we have not encountered a transformation event in >100 mice infused with MSC manipulated with transposons, we speculated that mutation by transposition was the inciting event. None of the identifiable transposition events occurred in a known proto-oncogene or tumor suppressor gene. This does not discount the possibility of insertional mutagenesis as the genomic lesion may have occurred on the chromosome which was subsequently disrupted or lost. Alternatively, genomic instability could have been a result of spontaneous unrepaired chromosomal lesion(s) that preceded the transposon insertion and resulted in osteosarcoma. These findings provide evidence of evolution of MSCs with osteogenic capacity into osteosarcoma in vivo and are clinically relevant as they document the potential of ex vivo manipulated MSCs for transformation into malignant disease.

EBV-PTLD is a known complication of alternative donor hematopoietic stem cell transplantation (HSCT) particularly when used in combination with HSC T-cell depletion and ATG. Initial reports in the setting of UCBT suggested a low incidence of EBV-PTLD (2–5%). We reviewed 335 consecutive patients (pts) who underwent UCBT at our institution between 7/19/94 and 3/29/05. Median age, weight, and follow-up were 16 years (0.2–69), 53.7kg (3.8–134.0) and 1.2 years (77 days9.2 years), respectively. Equine ATG (ATGAM) was used as part of a myeloablative (MA, Cy 120 mg/kg and TBI 1320–1375 cGy ± Flu 75 mg/m2) and non-myeloablative (NMA, Cy 50 mg/kg, Flu 200 mg/m2 and TBI 200 cGy) preparative regimen in 175/240 and 30/95 patients, respectively. As previously reported the overall incidence of EBV viremia and EBV-PTLD remained low. Overall, use of ATG lead to an increase in the incidence of EBV viremia (defined as > 1,000 copies of EBV DNA per mL of whole blood) and EBV-PTLD (14/205 [7%] with vs. 1/130 [0.7%] without ATG [p=0.16]), but this was not statistically significant. In recipients of a MA preparative regimen, EBV viremia or EBV-PTLD was observed in 8/175 (5%) who received ATG and in 0/65 who did not (p=0.81). However, a higher risk of EBV viremia and PTLD was observed in recipients of ATG and a NMA preparative regimen. EBV viremia or EBV-PTLD after NMA preparative regimen was observed in 6/30 (20%) pts who received ATG and 1/65 (2%) who did not (p

Mucopolysaccharidosis type IH (Hurler syndrome) is caused by alpha-L-iduronidase (IDUA) enzyme deficiency resulting in the accumulation of glycosaminoglycans (GAGs). This affects the function of many vital organs, causes mental retardation and early death. Although BMT can reduce many of these manifestations, pulmonary complications have limited the success of allogeneic BMT for Hurler patients. In our BMT population, non-infectious post-BMT lung complications were seen in 52% of MPS (n=23) vs 8% of patients with other metabolic storage diseases (n=25) (p To understand how IDUA deficiency affects lung function pre- and post-BMT, we examined the lungs of IDUA−/− mice (C57BL/6 background) and compared them to C57BL/6 wild type controls (WT). Alcian blue staining of lung cryosections prepared from 7–10 mo old IDUA−/− mice showed large areas of GAG deposition. These areas were associated with large perivascular and peribronchiolar infiltrates composed of predominantly CD4+ T cells with CD8+ T cells, CD11b+ cells and high numbers of MHC class II+ cells. Since GAGS have been shown in vitro to stimulate cells of the immune system, the co-localization of T cells, macrophages and accumulated GAGS sets the stage for the lung being a target injured by immune-mediated mechanisms. Pro-inflammatory cytokines (TNFα, IL-1β, IL-6, IL-12p70) were elevated (p

MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion>> Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.

MAPC are non-hematopoietic stem cells with the capacity to form most, if not all, cell types of the body. To date, the observations of homing of the MAPC have been limited to post mortem analyses. As MAPC may be useful in cellular therapies, our goal was to map their biodistribution in live organisms. To determine the real-time organ-specific homing pattern of donor MAPC, MAPC (from BM of C57BL/6J-rosa26 mice) were co-nucleofected with cDNAs encoding the red fluorescent protein DsRed2 and luciferase, using the Sleeping Beauty (SB) transposon system. Non-viral gene transfer mediated by SB is potentially advantageous to viral gene transfer because transposons may be less immunogenic since no viral proteins are present, and they are relatively easy to produce. DsRed2 and luciferase genes were cloned into plasmid vectors containing the transposase recognition sequences flanking the reporter genes (pT/CAGGS-DsRed2; pT/CAGGS-Luciferase). MAPC (106) were co-nucleofected (Amaxa, setting T-20, buffer T) with 5mcg of each marker plasmid and the SB transposase plasmid (p/CMV-HSB2) at a 1:50 ratio. 19% of MAPC expressed DsRed2 7 days after nucleofection. The MAPC were FACS sorted (1 cell per well) for cells with the highest DsRed2 expression. All MAPC tested expressed both DsRed2 and luciferase, suggesting that co-nucleofection is an efficient means of delivery of two plasmids. Two transgenic MAPC clones selected for further analysis were confirmed to be euploid by cytogenetic analysis, and maintained differentiation potential into the three germ layers. To verify transgene integration by transposition, the genomic sites of transposon integration were determined using splinkerette PCR. In the genome of MAPC clone 1, DsRed2 transposed in two sites on chromosome 5. One integration site (5qA3) was in the 3′ untranslated region of activin receptor interacting protein 1 (Acvrinp1). In clone 2 DsRed2 transposed into a single site on chromosome 10, in an intron of a gene termed SHPRH, which encodes a putative protein with SNF2/helicase and PHD-finger domains. To investigate the real time kinetics of MAPC population after infusion, 5 x 106 DsRed2 and luciferase positive MAPC (clone 2) were infused via tail vein into 8-week-old Rag2/IL-2Rgc−/− mice (T-, B- and NK-immunodeficient mice were used as a recipient to minimize the likelihood that the host would reject donor MAPC). Using whole body imaging (Xenogen) we were able to follow the distribution of the luciferase-marked MAPC over a period of 10 weeks. In addition, using DsRed2 expression the donor MAPC-derived cells in whole lung and in lung cryosections were identified. In summary, we show for the first time stable gene expression in adult stem cells using Sleeping Beauty transposon mediated non-viral gene transfer. These results show that MAPC-based cellular therapies can be monitored in vivo and suggest that transposon-based technology may be an attractive alternative to viral based gene delivery and therapy.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.

Finding disease markers (classifiers) from gene expression data by machine learning algorithms is characterized by a high risk of overfitting the data due the abundance of attributes (simultaneously measured gene expression values) and shortage of available examples (observations). To avoid this pitfall and achieve predictor robustness, state-of-the-art approaches construct complex classifiers that combine relatively weak contributions of up to thousands of genes (attributes) to classify a disease. The complexity of such classifiers limits their transparency and consequently the biological insights they can provide. The goal of this study is to apply to this domain the methodology of constructing simple yet robust logic-based classifiers amenable to direct expert interpretation. On two well-known, publicly available gene expression classification problems, the paper shows the feasibility of this approach, employing a recently developed subgroup discovery methodology. Some of the discovered classifiers allow for novel biological interpretations.

Gene Ontology (GO) terms are often used to interpret the results of microarray experiments. The most common approach is to perform Fisher’s exact tests to find gene sets annotated by GO terms which are over-represented among the genes declared to be differentially expressed in the analysis of microarray data. Another way is to apply Gene Set Enrichment Analysis (GSEA) that uses predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. However, after correcting for multiple hypotheses testing, few (or no) GO terms may meet the threshold for statistical significance, because the relevant biological differences are small relative to the noise inherent to the microarray technology. In addition to the individual GO terms, we propose testing of gene sets constructed as intersections of GO terms, Kyoto Encyclopedia of Genes and Genomes Orthology (KO) terms, and gene sets constructed by using gene–gene interaction data obtained from the ENTREZ database. Our method finds gene sets that are significantly over-represented among differentially expressed genes which cannot be found by the standard enrichment testing methods applied on individual GO and KO terms, thus improving the enrichment analysis of microarray data.

In this issue of a Blood , in a first-rate example of collaboration between academia (University of California) and industry (Sangamo), [Hoban et al][1] show in situ gene correction of sickle cell anemia (SCA), a prototypical hemoglobinopathy.[1][2] ![Figure][3] The peak of healthy

We contribute a novel, ball-histogram approach to DNA-binding propensity prediction of proteins. Unlike state-of-the-art methods based on constructing an ad-hoc set of features describing physicochemical properties of the proteins, the ball-histogram technique enables a systematic, Monte-Carlo exploration of the spatial distribution of amino acids complying with automatically selected properties. This exploration yields a model for the prediction of DNA binding propensity. We validate our method in prediction experiments, improving on state-of-the-art accuracies. Moreover, our method also provides interpretable features involving spatial distributions of selected amino acids.

Infusion of in vitro–derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant aids the recovery of the thymus damaged by total body irradiation. To understand the interaction between proTs and the thymic microenvironment, WT mice were lethally irradiated and given T cell–deficient (Rag1-/-) marrow with WT in vitro–generated proTs, limiting mature T cell development to infused proTs. ProTs within the host thymus led to a significant increase in thymic epithelial cells (TECs) by day 21 after transplant, increasing actively cycling TECs. Upon thymus egress (day 28), proT TEC effects were lost, suggesting that continued signaling from proTs is required to sustain TEC cycling and cellularity. Thymocytes increased significantly by day 21, followed by a significant improvement in mature T cell numbers in the periphery by day 35. This protective surge was temporary, receding by day 60. Double-negative 2 (DN2) proTs selectively increased thymocyte number, while DN3 proTs preferentially increased TECs and T cells in the spleen that persisted at day 60. These findings highlight the importance of the interaction between proTs and TECs in the proliferation and survival of TECs and that the maturation stage of proTs has unique effects on thymopoiesis and peripheral T cell recovery.

This paper presents a method that uses gene ontologies, together with the paradigm of relational subgroup discovery, to help find description of groups of genes differentially expressed in specific cancers. The descriptions are represented by means of relational features, extracted from gene ontology information, and are straightforwardly interpretable by the medical experts. We applied the proposed method to two known data sets: acute lymphoblastic leukemia (ALL) vs. acute myeloid leukemia and classification of fourteen types of cancer. Significant number of discovered groups of genes had a description, confirmed by the medical expert, which highlighted the underlying biological process that is responsible for distinguishing one class from the other classes. We view our methodology not just as a prototypical example of applying sophisticated machine learning algorithms to microarray data, but also as a motivation for developing more sophisticated functional annotations and ontologies, that can be processed by such learning algorithms.