Lentiviral vectors pseudotyped with glycoproteins from Ross River and vesicular stomatitis viruses: variable transduction related to cell type and culture conditions

HIV-1-derived lentiviral vectors have been pseudotyped with various envelope glycoproteins to alter their host range. Previously, we found that envelope glycoproteins derived from the alphavirus Ross River virus (RRV) can pseudotype lentiviral vectors and mediate efficient transduction of a variety of epithelial and fibroblast-derived cell lines. In this study, we have investigated transduction of hematopoietic cells using RRV-pseudotyped vectors encoding the enhanced green fluorescent protein (EGFP). RRV-mediated transduction of human CD34+ cord blood cells and progenitors was very inefficient, even at multiplicities of infection of 100 (0.4% EGFP-positive progenitor colonies). Inefficient transduction was also observed in a variety of hematopoietic cell lines. However, two erythroleukemia-derived cell lines and monocytic cells that were driven to macrophage-like differentiation were moderately transduced. Transduction of hematopoietic cells with a control VSV-G-pseudotyped lentiviral vector was generally efficient, but unexpectedly decreased up to threefold upon stimulation of lymphocytic cell lines or primary murine bone marrow cells. Also, the tested hematopoietic cell lines were essentially nonpermissive for adeno-associated type 2 (AAV) vectors, and this was not affected by lineage, activity, or differentiation. Treatment of permissive 293 cells with proteases revealed that transduction with both the RRV- and the VSV-G-pseudotyped vectors in part depends on the presence of cell surface proteins. These results show a severely restricted ability of RRV glycoproteins to mediate transduction in hematopoietic cells that is likely due to specific receptor requirements that differ from those of VSV-G and AAV. Conversely, transduction with the VSV glycoprotein is affected by cellular activation more than widely believed. Our findings suggest that the envelope glycoproteins and culture conditions employed need to be carefully evaluated for each application. Furthermore, the uniquely restricted host range of RRV-pseudotyped vectors may aid in the design of novel cell-selective transduction strategies

Sequential dissociation of ionized benzonitrile: New pathways to reactive interstellar ions and neutrals

Since benzonitrile’s discovery in the interstellar medium (ISM) in 2018, several studies have explored the strongest unimolecular dissociations of its radical cation (C6H5CN•+). However, sequential dissociation processes, which become important when ionization occurs with significant excess energy transfer, have received almost no attention to date. The present metastable dissociative ionization experiments reveal 14 different dissociations, of which 11 have never been observed before. Nine of these new reactions involve the dissociation of a fragment ion. A notable result shows that C4H2 •+ production (the second most intense fragment ion in conventional mass spectra without metastable dissociation analysis) derives from sequential dissociation via C6H4 •+ , as well as from the previously reported unimolecular dissociation of C6H5CN •+ . Furthermore, our experiments demonstrate new pathways that produce astrochemically important neutrals including HCN/CNH and CN• , as well as revealing CH• and C3H• production from ionized benzonitrile for the first time. In addition to the metastable dissociation experiments, we applied density functional theory to calculate two sequential dissociation routes and report the results of our detailed analysis of the peak shapes in a conventional mass spectrum of benzonitrile. The latter enabled the dominant ion to be identified in peaks with nearest-integer m/z values that match two conceivable ions. The present identification of C6H2N+ production using this approach allows its presence in the ISM to be inferred for the first time. This paper extends our understanding of how the dissociative ionization of benzonitrile can contribute to the abundances of radicals and other reactive species in interstellar environments

CELLULAR THERAPY AND HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR CANCER

poster abstractThe Center for Cellular Therapy and Hematopoietic Stem Cell Transplantation for Cancer was established in July 2007 to promote translational and clinical research in cellular therapy for cancer. The primary goal of the Center is translate discoveries from bench-to-clinic through phase I and early phase II cellular therapy clinical trials. To achieve this objective, the Center has brought together the unique expertise in hematopoiesis, immunology, gene therapy, graft engineering, and clinical hematopoietic stem cell transplantation (HCT) available at IUPUI. Since its establishment, we have completed two phase I clinical trials developing novel preparative regimens for allogeneic and autologous stem cell transplantation for patients with refractory leukemia and lymphoma, respectively. In addition, we have also initiated 5 additional early phase clinical trials that directly translate IUPUI laboratory discoveries to patients with hematological cancers. The Center has successfully competed for external funding through peerreviewed grants and pharmaceutical contracts. In this presentation, we highlight some important examples of the Center’s ongoing and completed research. An important clinical research focus of our Center is the ability to extend the curative potential of allogeneic HCT to patients without suitably HLA-matched donors. We are currently exploring ways to improve the outcomes of umbilical cord blood (UCB) and haplotype-mismatched stem cell transplantation for patients with hematological cancers. The discovery in Dr. Broxmeyer’s Laboratory, Indiana University, Indianapolis, that inhibition of the enzyme CD26 promotes homing and engraftment of limiting numbers of UCB stem cells has been translated to the first clinical trial in vivo CD26 inhibition using sitagliptin in adult leukemia patients undergoing UCB transplantation. Our preliminary data indicates that high-dose sitagliptin is well tolerated and appears to shorten the time of engraftment. As our data is further confirmed in this pilot study, we plan to investigate this potentially paradigm changing approach in a larger national study. As an extension of this research, Dr. Pelus’ Laboratory, Indiana University, Indianapolis, has shown that short-term ex vivo treatment of hematopoietic progenitors using PGE2 will also promote engraftment. We are currently investigating the potential synergy of PGE2 treatment with CD26 inhibition to further enhance engraftment, which if results appear promising will also be translated to a phase I clinical trial. In haplotypemismatched allogeneic HCT, mismatching of donor KIR receptors on natural killer (NK) cells with recipient KIR ligands expressed on the patient’s tumor cells exerts a NK cell-mediated antileukemia effect that contributes to reduced relapse after transplantation. We (Dr. Farag’s Laboratory, Indiana University, Indianapolis) have shown that in vivo donor derived NK cells developing from donor stem cells have an “inhibitory” receptor phenotype that may suboptimally function against leukemia. This has resulted in a phase I trial of purified NK cell infusion following mismatched HCT to investigate the feasibility and safety of this approach, as a prelude to a larger study to investigate its efficacy. Although the highest dose level of NK cells has not yet been investigated, the preliminary data indicates that such a novel approach is feasible. In additional studies based on our laboratory findings, we are exploring the harnessing of NK cells in the therapy of cancer through the monoclonal antibodies that block KIR receptors in combination with immuno-modulatory agents (e.g., lenalidomide) and antibodies that promote antibody-dependent cellular cytotoxicity (e.g., rituximab, anti-CS1). We have initiated patents for these discoveries, and are currently planning to transplant these into phase I clinical trials. Other ongoing research includes enhancing immune function against cancer through STAT3 inhibition to overcome tumor-mediated impairment of dendritic cell maturation, ex vivo specific expansion of cytotoxic of NK cell subsets for clinical use, and enhancing immune cell function following transplantation. The continued success of our Center will depend on a continuing pipeline of novel laboratory discoveries and their translation to early phase clinical trials to assess feasibility and safety as a prelude to larger trials assessing efficacy. Initial funding of the Center by IUPUI has allowed the Center’s conception, and the bringing together of basic and clinical researchers to the “research table” to make this translational/clinical research endeavor a reality, and has allowed us to be competitive for external funding. An important developing outcome of this initiative is the preparation for a Program Project grant in Mobilization and Engraftment of Stem Cells

Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy

Abrogated cryptic activation of lentiviral transfer vectors

Despite significant improvements in lentivirus (LV) vector-based gene therapy there are still several safety risks using LV vectors including the potential formation of replication-competent LV particles. To address this shortcoming, we constructed a novel and safer gene transfer system using modified SIN-based LV gene transfer vectors. Central to our approach is a conditional deletion of the Ψ packaging signal after integration in the target genome. Here we demonstrate that after transduction of target cells, conventional SIN-based LV transfer vectors can still be mobilized. However mobilization is rendered undetectable if transductions are followed by a Cre/loxP-mediated excision of Ψ. Thus conditional elimination of the packaging signal may represent another advance in increasing the safety of LV vectors for gene therapeutic treatment of chronic diseases

The Access Technology Program of the Indiana Clinical Translational Sciences Institute (CTSI): A model to facilitate access to cutting-edge technologies across a state

Introduction: Access to cutting-edge technologies is essential for investigators to advance translational research. The Indiana Clinical and Translational Sciences Institute (CTSI) spans three major and preeminent universities, four large academic campuses across the state of Indiana, and is mandate to provide best practices to a whole state. Methods: To address the need to facilitate the availability of innovative technologies to its investigators, the Indiana CTSI implemented the Access Technology Program (ATP). The activities of the ATP, or any program of the Indiana CTSI, are challenged to connect technologies and investigators on the multiple Indiana CTSI campuses by the geographical distances between campuses (1–4 hr driving time). Results: Herein, we describe the initiatives developed by the ATP to increase the availability of state-of-the-art technologies to its investigators on all Indiana CTSI campuses, and the methods developed by the ATP to bridge the distance between campuses, technologies, and investigators for the advancement of clinical translational research. Conclusions: The methods and practices described in this publication may inform other approaches to enhance translational research, dissemination, and usage of innovative technologies by translational investigators, especially when distance or multi-campus cultural differences are factors to efficient application

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. Haemophilia is a genetic bleeding disorder associated with a deficiency in the coagulation factor VIII. Here, the authors use gene therapy to achieve stable overexpression of factor VIII in platelets of dogs with haemophilia A, preventing the occurrence of severe bleeding episodes for over 2.5 years