Production of Lentivirus for the Establishment of CAR-T Cells.

Production of Lentivirus for the Establishment of CAR-T Cells. Methods Mol Biol. 2020;2086:61-67 Authors: Lana MG, Strauss BE Abstract One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the...
Source: Mol Biol Cell - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research

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Reena Goswami1, Gayatri Subramanian2, Liliya Silayeva1, Isabelle Newkirk1, Deborah Doctor1, Karan Chawla2, Saurabh Chattopadhyay2, Dhyan Chandra3, Nageswararao Chilukuri1 and Venkaiah Betapudi1,4* 1Neuroscience Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen, MD, United States 2Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, United States 3Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States 4Department of Physiology and Biophysics, Case Western Reserve University, Clev...
Source: Frontiers in Oncology - Category: Cancer & Oncology Source Type: research
Discussion In this section, we discuss the mechanisms responsible for lymphomagenesis in the various inborn errors of immunity and provide an overview of the treatment. Defects in Immune Responses That Predispose to Lymphomagenesis in PIDDs The complex immune mechanisms and their interplay that predisposes to neoplastic transformation of B or T cells and development of lymphomas in PIDD patients has not been fully elucidated. However, it is expected that the etiology in most cases is multifactorial and related to a dynamic regulation of immune response and environmental triggers (Figure 3). An underlying intrinsic susce...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research
Discussion Suppressor of cytokine signaling 1 is an essential molecule for maintaining immune homeostasis and subverting inflammation. Disorders arising from excess inflammation or SOCS1 deficiency can be potentially treated with SOCS1 mimetics (Ahmed et al., 2015). While SOCS1 has promising potential in many disorders, it should be noted that new targets and actions of SOCS1 are still being discovered and not all the effects of this protein are beneficial in autoimmune diseases and cancer. For instance, SOCS1 degrades IRS1 and IRS2, required for insulin signaling, via the SOCS Box domain, thus, limiting its potential in ...
Source: Frontiers in Pharmacology - Category: Drugs & Pharmacology Source Type: research
Conclusions: CAR T cell therapies have demonstrated the clinical benefits of harnessing our body's own defenses to combat tumor cells. Similar research is being conducted on lesser known modifications and gene-modified immune cells, which we highlight in this review. Introduction Chimeric antigen receptors and engineered T cell receptors (based on previously identified high affinity T cell receptors) function by redirecting T cells to a predefined tumor-specific (or tumor-associated) target. Most of these modifications use retroviral or lentiviral vectors to integrate the construct, and most of the receptors ...
Source: Frontiers in Oncology - Category: Cancer & Oncology Source Type: research
In this study, we show that calorie restriction is protective against age-related increases in senescence and microglia activation and pro-inflammatory cytokine expression in an animal model of aging. Further, these protective effects mitigated age-related decline in neuroblast and neuronal production, and enhanced olfactory memory performance, a behavioral index of neurogenesis in the SVZ. Our results support the concept that calorie restriction might be an effective anti-aging intervention in the context of healthy brain aging. Greater Modest Activity in Late Life Correlates with Lower Incidence of Dementia ...
Source: Fight Aging! - Category: Research Authors: Tags: Newsletters Source Type: blogs
For more than twenty years now, Richard Burt's research teams have been working on the treatment of autoimmunity through the destruction and recreation of the immune system. Autoimmunity is a malfunction in the self-tolerance of immune cells, leading them to attack patient tissues. The malfunction is entirely contained in the immune system, so if the immune system is destroyed and replaced, the autoimmunity stops. If the genesis of autoimmunity is happenstance, an unfortunate one-time accident, then this is a cure. But if autoimmunity has a trigger outside the immune system in a given patient, it will return after some per...
Source: Fight Aging! - Category: Research Authors: Tags: Medicine, Biotech, Research Source Type: blogs
In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplic...
Source: Blood - Category: Hematology Authors: Tags: 801. Gene Therapy and Transfer: Poster II Source Type: research
Autologous CD19 directed CAR T-cell therapy has response rates of>70% in adult acute lymphoblastic leukemia (ALL) and>40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 &CD22 with intracellular signaling domains inco...
Source: Blood - Category: Hematology Authors: Tags: 801. Gene Therapy and Transfer: Clinical Trials for Hemophilia and Using CAR T Cells Source Type: research
We report that the disruption of excitation-contraction coupling contributes to impaired force generation in the mouse model of Sod1 deficiency. Briefly, we found a significant reduction in sarcoplasmic reticulum Ca2+ ATPase (SERCA) activity as well as reduced expression of proteins involved in calcium release and force generation. Another potential factor involved in EC uncoupling in Sod1-/- mice is oxidative damage to proteins involved in the contractile response. In summary, this study provides strong support for the coupling between increased oxidative stress and disruption of cellular excitation contraction mac...
Source: Fight Aging! - Category: Research Authors: Tags: Newsletters Source Type: blogs
Curry J, Featherston S, Foglesong J, Shoberu B, Gulbis A, Mireles ME, Hafemeister L, Nguyen C, Kapoor N, Rezvani K, Neelapu SS, Shpall EJ, Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network Abstract In 2017, an autologous chimeric antigen receptor (CAR) T cell therapy indicated for children and young adults with relapsed and/or refractory CD19+ acute lymphoblastic leukaemia became the first gene therapy to be approved in the USA. This innovative form of cellular immunotherapy has been associated with remarkable response rates but is also associated with unique and often severe toxicities, which ...
Source: Clinical Lymphoma and Myeloma - Category: Cancer & Oncology Authors: Tags: Nat Rev Clin Oncol Source Type: research
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