Engineering CAR-T Cells for Improved Function Against Solid Tumors

Genetic engineering T cells to create clinically applied chimeric antigen receptor (CAR) T cells has led to improved patient outcomes for some forms of hematopoietic malignancies. While this has inspired the biomedical community to develop similar strategies to treat solid tumor patients, challenges such as the immunosuppressive character of the tumor microenvironment, CAR-T cell persistence and trafficking to the tumor seem to limit CAR-T cell efficacy in solid cancers. This review provides an overview of mechanisms that tumors exploit to evade eradication by CAR-T cells as well as emerging approaches that incorporate genetic engineering technologies to improve CAR-T cell activity against solid tumors

Why Are Students Not Majoring in Information Systems?

The purpose of this study was to examine some of the factors that influence and impact business students when they select their major and, more particularly, to examine why students are not majoring in information systems. Students in an entry level business class responded that they were more knowledgeable about careers in management, marketing, accounting, and finance than they were about careers in information systems. These business students indicated that they are looking for majors that will be interesting, provide them with job security initially and over their careers, and pay them well. The most important information sources used by these students in their major selection decision were information on college/department websites, brochures about the major, and information on the Internet. When asked why they were not majoring in information systems, the top two reasons given were not what I wanted to do and subject not of interest

Perforin gene transfer into hematopoietic stem cells improves immune dysregulation in murine models of perforin deficiency

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8 + T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8 + lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL

Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells

Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral LTR-driven vectors, which semi-randomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSC) and can impede transgene expression during differentiation. In this study, we aimed to correct p47phox-deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47phox transgene into the AAVS1 locus of human induced PSC (iPSC) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes and MRP8). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoter achieved therapeutic-relevant levels of p47phox expression and NADPH oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells

Biosafety Studies of a Clinically Applicable Lentiviral Vector for the Gene Therapy of Artemis-SCID

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic in vitro or in vivo. Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. In vitro, the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID

Maximum Azimuthal Anisotropy of Neutrons from Nb-Nb Collisions at 400 AMeV and the Nuclear Equation of State

We measured the first azimuthal distributions of triple--differential cross sections of neutrons emitted in heavy-ion collisions, and compared their maximum azimuthal anisotropy ratios with Boltzmann--Uehling--Uhlenbeck (BUU) calculations with a momentum-dependent interaction. The BUU calculations agree with the triple- and double-differential cross sections for positive rapidity neutrons emitted at polar angles from 7 to 27 degrees; however, the maximum azimuthal anisotropy ratio for these free neutrons is insensitive to the size of the nuclear incompressibility modulus K characterizing the nuclear matter equation of state.Comment: Typeset using ReVTeX, with 3 ps figs., uuencoded and appende

Neutrons from multiplicity-selected Au-Au collisions at 150, 250, 400, and 650 AMeV

We measured neutron triple-differential cross sections from multiplicity-selected Au-Au collisions at 150, 250, 400, and 650 \AMeV. The reaction plane for each collision was estimated from the summed transverse velocity vector of the charged fragments emitted in the collision. We examined the azimuthal distribution of the triple-differential cross sections as a function of the polar angle and the neutron rapidity. We extracted the average in--plane transverse momentum $\langle P_x\rangle$ and the normalized observable $\langle P_x/P_\perp\rangle$, where $P_\perp$ is the neutron transverse momentum, as a function of the neutron center-of-mass rapidity, and we examined the dependence of these observables on beam energy. These collective flow observables for neutrons, which are consistent with those of protons plus bound nucleons from the Plastic Ball Group, agree with the Boltzmann--Uehling--Uhlenbeck (BUU) calculations with a momentum--dependent interaction. Also, we calculated the polar-angle-integrated maximum azimuthal anisotropy ratio R from the value of $\langle P_x/P_\perp\rangle$.Comment: 20 LaTeX pages. 11 figures to be faxed on request, send email to sender's addres

Neutrons from multiplicity-selected La-La and Nb-Nb collisions at 400A MeV and La-La collisions at 250A MeV

Triple-differential cross sections for neutrons from high-multiplicity La-La collisions at 250 and 400 MeV per nucleon and Nb-Nb collisions at 400 MeV per nucleon were measured at several polar angles as a function of the azimuthal angle with respect to the reaction plane of the collision. The reaction plane was determined by a transverse-velocity method with the capability of identifying charged-particles with Z=1, Z=2, and Z > 2. The flow of neutrons was extracted from the slope at mid-rapidity of the curve of the average in-plane momentum vs the center-of-mass rapidity. The squeeze-out of the participant neutrons was observed in a direction normal to the reaction plane in the normalized momentum coordinates in the center-of-mass system. Experimental results of the neutron squeeze-out were compared with BUU calculations. The polar-angle dependence of the maximum azimuthal anisotropy ratio $r(\theta)$ was found to be insensitive to the mass of the colliding nuclei and the beam energy. Comparison of the observed polar-angle dependence of the maximum azimuthal anisotropy ratio $r(\theta)$ with BUU calculations for free neutrons revealed that $r(\theta)$ is insensitive also to the incompressibility modulus in the nuclear equation of state.Comment: ReVTeX, 16 pages, 17 figures. To be published in Physical Review