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(2017). A Model for Tissue Banking in the Community Setting. Oncology Issues: Vol. 32, No. 4, pp. 36-48.

Background: Allogeneic HSCT from either an HLA-identical sibling or an unrelated donor is a potentially curative treatment for patients with hemoglobinopathies and erythroid disorders (ED), such as Thalassemia Major (TM), Sickle Cell Disease (SCD) and Diamond-Blackfan Anemia (DBA). The limited historical experience with HLA-haploidentical HSCT in this setting has reported a disease-free survival probability lower than that reported using HLA-matched donors. In the last few years, we have developed a novel method of graft manipulation, based on the selective depletion of α/β+ T-cells and CD19+ B-cells (ClinicalTrial.gov identifier: NCT01810120), which was shown to be safe and effective in children with multiple types of non-malignant disorders (Bertaina el al, Blood 2014). To further optimize this approach through the acceleration of the recovery of adaptive immunity, we designed an ongoing phase I/II trial aimed to test the safety and efficacy of post-transplant infusion of donor T-cells transduced with the iC9 suicide gene (BPX-501 cells) in children with either malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). As the transduced gene contains sequences for the CD19 marker, BPX-501 cells (CD3+/CD19+) can be easily tracked in peripheral blood. We report on 10 children with hemoglobinopathies and ED who were enrolled in the phase II portion of the study. Patients and methods: Five patients were males and 5 were females, and median age at diagnosis and at HSCT was 5.34 and 9.52 years (range 2.33-11.71), respectively. Seven patients had TM (all bo/bo), 2 DBA and 1 SCD. All 10 patients were transfusion-dependent and receiving iron-chelation therapy before haplo-HSCT. Among the thalassemia patients, 4 patients belonged to class I and 3 to class II of the Pesaro classification. All patients were transplanted from a parent. Median number of CD34+ and αβ+ T-cells infused was 22.5 x 106/kg and 0.3 x 105/kg, respectively. In all patients, conditioning regimen included busulfan (16 mg/Kg), thiotepa (10 mg/Kg) and fludarabine (160 mg/m2). Rabbit ATG (12 mg/Kg over 3 days, from day -4 to day -2) was administered to prevent graft-versus-host disease (GvHD) and graft failure and Rituximab (200 mg/ m2 on day -1) was administered to prevent EBV-related lymphoproliferative disorders. No post-transplantation GvHD prophylaxis was given. Median follow-up is 301 days (range 41-420 days). Basic phenotyping of circulating lymphocytes was assessed by flow cytometry on fresh heparinized peripheral blood samples at 10, 20, 30, 60, 90, 120 and 150 days post haplo-HSCT. Results: After haplo-HSCT, the median time to reach neutrophil and platelet recovery was 14 days (range 11-28) and 10 days (range 8-12), respectively. After engraftment of the allograft, BPX-501 cells were infused (dose: 1x106 cells/kg) at a median time of 13.5 days after HSCT (range 10-26). Nine of the 10 patients maintained sustained donor engraftment, reaching full chimerism. The patient who experienced secondary graft failure was successfully re-transplanted from the same parent and he is full donor chimeric with transfusion-independence. Grade I/II skin acute GvHD occurred in 2 patients (at 31 and 59 days after HSCT, respectively). There was no occurrence of chronic GVHD. Remarkably, no patient has died and none of the patients have been re-hospitalized after initial discharge. The last erythrocyte transfusion was administered on day +7 post-transplant (range 4-33 days). At last follow-up, the median hemoglobin value of these patients was 11.35 gr/dL (range 10.2-13.4). BPX-501 cells expanded after infusion and still persist in all patients at last follow-up. All children are alive and transfusion-independent. Details on T cell, NK cell and B cell recovery are shown in Figure 1 (Panel A-D). Conclusions: Children with hemoglobinopathies and DBA can benefit from curative haplo-HSCT after depletion of α/β T-cells followed by infusion of BPX-501 cells, which, expanding and persisting over time, contribute to speed immune recovery of adaptive T-cell immunity, thus rendering the procedure safer. Figure 1 Figure 1. Disclosures French: Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy. In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods. Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice. Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP). The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg. The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter. The average magnitude of induction was 14- to 19-fold. Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated. In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product. In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks. Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications.

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of angiostatic factors should be a viable approach for cancer gene therapy. Endostatin, a potent angiostatic factor, was expressed in mouse muscle and secreted into the bloodstream for up to 2 weeks after a single intramuscular administration of the endostatin gene. The biological activity of the expressed endostatin was demonstrated by its ability to inhibit systemic angiogenesis. Moreover, the sustained production of endostatin by intramuscular gene therapy inhibited both the growth of primary tumors and the development of metastatic lesions. These results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers.

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.

We investigated the ability of an improved mifepristone-dependent GeneSwitch system to regulate the expression of genes for two therapeutic proteins: vascular endothelial growth factor (VEGF) and erythropoietin. The GeneSwitch system consisted of two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene. When the constitutive CMV promoter of the GeneSwitch plasmid was replaced by an autoinducible promoter consisting of four copies of GAL4 DNA binding sites linked to a minimal thymidine kinase promoter, the tightness of transgene regulation was improved by an order of magnitude. Quantitative RT-PCR analysis of GeneSwitch mRNA confirmed that the autoinducible promoter was responsive to mifepristone. We demonstrated the ability of the improved GeneSwitch system to regulate the expression of VEGF or erythropoietin in a biologically relevant manner after delivery of plasmids to the hind-limb muscle of adult mice. This ability of the autoinducible GeneSwitch system to regulate the expression of therapeutic proteins in mice indicates its potential for use in human gene therapy applications.

A muscle-specific gene medicine is described that provides for long-term secretion of biologically active human growth hormone (hGH) from skeletal muscle into the systemic circulation. The hGH gene medicine is composed of a muscle-specific hGH plasmid expression system complexed with a protective, interactive, non-condensing (PINC) delivery system. The muscle-specific gene expression system, pSK-hGH-GH, was constructed by linking the promoter/enhancer regions of chicken skeletal alpha-actin to hGH gene. C2C12 myoblast transfection with pSK-hGH-GH resulted in the synthesis of hGH in a muscle-specific manner. Direct injection into rat tibialis cranialis muscle of pSK-hGH-GH complexed with a polymeric PINC delivery system, polyvinylpyrrolidone (PVP), produced hGH levels in muscle that were 10- to 15-fold higher compared with plasmid formulated in saline at 14 days post-injection. Intratracheal instillation in rat lung of pSK-hGH-GH did not produce significantly detectable levels of hGH. In hypophysectomized rats, a single intramuscular dose of the pSK-hGH-GH/PVP complex resulted in hGH expression and a subsequent increase in serum levels of rat IGF-I and growth. hGH expression and effects on rat serum IGF-I levels were detectable up to 28 days after injection of formulated plasmid and effects on growth were detectable unto 21 days. Anti-hGH antibodies were detectable in serum at 14 days post-injection, reached a plateau at 21 days, and remained elevated through the study period. Cyclosporin treatment of the pSK-hGH-GH/PVP-injected animals completely inhibited the antibody response and resulted in increased hGH expression.

Recent evidence has shown that insulin-like growth factor-I (IGF-I) plays an important role in the development, maintenance, and regeneration of peripheral nerves and skeletal muscle. IGF-I offers the potential to treat neuromuscular diseases in humans. We have developed a nonviral gene therapy method to express and produce localized and sustained therapeutic levels of IGF-I within target muscles by intramuscular injection of formulated plasmids. The purpose of the present study was to demonstrate that intramuscular injection of a plasmid encoding human IGF-I (hIGF-I) and engineered to restrict expression to skeletal muscle produces sustained local concentrations of biologically active hIGF-I. Normal rats received a single intramuscular injection of plasmids formulated as a complex with polyvinylpyrrolidone (PVP). Results show that hIGF-I mRNA and hIGF-I protein were detectable in the injected muscles for the duration of the study (28 days), whereas the hIGF-I protein was not detected in blood. Biological activity of hIGF-I was determined by immunodetection of a nerve-specific growth-associated protein, GAP-43, an indicator of motor neuron sprouting. Placement of human growth hormone (hGH) 3' untranslated region enhanced GAP-43 staining, probably due to improved secretion of hIGF-I. Enhanced immunoreactivity of GAP-43 was observed in muscles injected with the formulated hIGF-I plasmid when compared to controls. These results demonstrate that intramuscular injection of hIGF-I plasmid formulated as a complex with PVP produces a localized and sustained level of biologically active hIGF-I.

The concentration of the iron-binding protein lactoferrin was measured in vaginal mucus of women throughout the menstrual cycle. Lactoferrin is proposed to limit growth of mucosal pathogens such as Neisseria gonorrhoeae. Lactoferrin concentration in mucus was highest just after menses (62.9 to 218 micrograms/mg of protein) and lowest (3.8 to 11.4 micrograms/mg of protein) just before menses. Overall concentration of mucus protein showed no variation with menstrual cycle, and little lactoferrin was detectable in cell debris. Plasma lactoferrin did not show the variation seen in vaginal mucus. Because of the suggestion of hormonal influence on vaginal lactoferrin, its concentration was determined in women receiving oral contraceptive therapy. Mean lactoferrin concentration in women taking oral contraceptives was significantly lower than in the control group (never exceeding 19.8 micrograms/mg of protein) and showed no monthly variation. Vaginal lactoferrin appears to be under hormonal control. Variation in vaginal lactoferrin concentration may result in alterations in susceptibility to bacterial pathogens such as Neisseriae gonorrhoeae.