To look for the average vector copy number (VCN) per cell, two qPCR reactions (optimized woodchuck hepatitis computer virus post-transcriptional regulatory element [oPRE] and FVII as research gene) were carried out. graft-versus-host disease model system that closely mimics haploidentical hematopoietic stem cell transplantation, an approach that is now being evaluated for use in the medical center. Control animals succumbed quickly to disease, whereas treatment with apceth-201 resulted in long-term survival of 57% of the animals. Within 25?days after the second injection, clinical scores returned to baseline in responding animals, indicating complete resolution of graft-versus-host disease. These encouraging data have led to planning of a phase I study using apceth-201. gene.19, 20 Under physiological conditions, AAT inactivates the serine protease elastase released from neutrophils during inflammation and prevents tissue damage from uncontrolled immune responses.21, 22 It has been shown that AAT inhibits the production of proinflammatory cytokines, such as interleukin-1beta (IL-1), tumor STING agonist-1 necrosis factor alpha (TNF-), and IL-8, by monocytes and peripheral blood mononuclear cells (PBMCs), while at the same time promoting the secretion of STING agonist-1 anti-inflammatory molecules, such as IL-10 and IL-1 receptor antagonist.23, 24, 25, 26, 27, 28, 29, 30 Furthermore, recombinant AAT has already been tested in clinical trials for GvHD with encouraging results.31, 32 Because of the promising anti-inflammatory properties of AAT and MSCs individually, we sought to combine these therapeutic modalities into a cell-based gene therapy product for the treatment of aGVHD. The apceth-201 product consists of human MSCs that have been lentivirally transduced to express and secrete AAT. The primary aim of the present study was to characterize apceth-201 and assess the therapeutic efficacy in pre-clinical murine models of GvHD. The secondary aim was to assess the security of apceth-201, specifically to determine whether immunomodulation by apceth-201 would also impair graft-versus-leukemia effects or T?cell-mediated antibody responses. Results Vector Design and Characterization of Transduced MSCs As the first step in generating AAT-producing MSCs, a lentiviral vector was designed. The pCCL backbone was a kind gift of Donald Kohn. The gene was inserted and placed under the control of an elongation factor (EF) short (s) promoter to achieve constitutive expression (Physique?1A).33 The encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) was included in the vector to ensure co-expression of the AAT transgene and a puromycin resistance cassette,34 and transduced cells were determined using puromycin. Open in a separate window Physique?1 Vector Design and Characterization of Transduced MSCs (A) Sequence of the AAT lentiviral vector: HIV, long STING agonist-1 terminal repeat (LTR), elongation factor alpha short promoter (EFs), AAT gene mode of action of apceth-201. Cell counts for reddish and white blood cells, as well as for platelets, were substantially improved in mice treated with MSCs, suggesting that short-term responses may not be AAT mediated (Physique?5B). Strikingly, bone marrow cellularity was significantly higher in mice treated with apceth-201 compared to those that received native MSCs (Physique?5C). The serum content of multiple cytokines was analyzed using a cytometric bead array assay. We found that production of two of the most crucial cytokines for mediating aGvHD responses, IFN and soluble TNF- receptor, were substantially reduced in mice treated either with native MSCs or apceth-201 (Physique?5D). The survival cohort was monitored daily to determine clinical scores based on pre-determined criteria (see the Materials and Methods; Physique?5E). Animals were removed from the study and humanely euthanized when they reached a cumulative score of 8. The median survival of untreated animals was 18?days, and it could be significantly prolonged to 22?days by treating mice with native MSCs (Physique?5F). Survival was further enhanced by administering apceth-201, which increased the median survival to 38 and 36?days, following the administration of low and high doses, respectively. Altogether, these data show that apceth-201 cells robustly guarded the cells of the bone marrow from targeted immune destruction. Furthermore, this translates into a long-term effect in the form of a significantly increased survival benefit, despite no obvious short-term differences in peripheral blood counts. Administering apceth-201 Attenuates Disease in an All-Murine Model of GvHD Having established that apceth-201 cells provide a significant survival benefit over native STING agonist-1 MSCs in a humanized model of GvHD, we utilized an all-murine model of aGvHD to Rabbit polyclonal to Smad2.The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene ‘mothers against decapentaplegic’ (Mad) and the C.elegans gene Sma. STING agonist-1 investigate the mechanism by which this protection may be conferred. The all-murine model is usually representative of haploidentical HSCT, the feasibility of which is currently being explored in the medical center.53 Splenocytes.