In Vivo Delivery of a CD20 CAR Using a CD8-Targeted Fusosome in Southern Pig-Tail Macaques (M. nemestrina) Results in B Cell Depletion

Introduction: Ex vivo manufactured chimeric antigen receptor (CAR) T cell therapies are highly effective for treating B cell malignancies. However, the complexity, cost and time required to manufacture CAR T cells limits access. To overcome conventional ex vivo CAR T limitations, a novel gene therapy platform has been developed that can deliver CAR transgenes directly to T cells through systemic administration of a fusosome, an engineered, target-directed novel paramyxovirus-based integrating vector that binds specific cell surface receptors for gene delivery through membrane fusion. Here, we demonstrate that systemic administration of a CD8a-targeted, integrating vector envelope (i.e., fusogen) encoding an anti-CD20 CAR into Southern pig-tail macaques (M. nemestrina), which is a species permissive to the integrating vector-mediated transduction, results in T cell transduction and B cell depletion with no treatment-related toxicities. Methods: CD8a-specific single chain variable fragments (scFvs) were generated and measured for target specificity versus non-CD8-expressing cells in vitro. Cross-reactivity of the CD8a-specific fusogen for human and nemestrina T cells was confirmed in vitro. Targeted fusogens were then used to pseudotype integrating vector expressing an anti-CD20 CAR containing the 4-1BB and CD3zeta signaling domains (CD8a-anti-CD20CAR). Transduction and B cell killing was confirmed on human and nemestrina PBMCs. To evaluate in vivo activity, normal, healthy nemestrina macaques were treated with a single dose of CD8a-targeted anti-CD20 CAR fusosome (n=6) or saline (n=2) via intravenous infusion at 10mL/kg/hr for 1-hour and evaluated for up to 52 days for evidence of adverse effects, B cell depletion, CAR-mediated cytokine production, CAR T cell persistence and vector biodistribution using ddPCR and anti-CD20CAR transgene by RT-ddPCR to detect transgene levels. Histopathology of several organs and immunohistochemistry for CD3 and CD20 on lymph nodes, spleen, and bone marrow were performed at termination (days 35 and 52). Tolerability of the treatment was assessed by body weight, body temperature, neurological exams, serum chemistry panel, and complete blood counts pre-dose and post-dose up to 52 days. Results: The CD8a-targeted fusogen demonstrated CD8a-specificity versus human CD8 negative cell lines, and cross-reactivity and transduction efficiency in nemestrina PBMCs in vitro. Compared to a control vector (GFP), anti-CD20CAR-modified T cells showed a dose-dependent depletion of B cells using in vitro assays. Following infusion of CD8a-anti-CD20CAR fusosomes into macaques, pharmacological activity in peripheral blood was detected by a reduction of B cells in 4 of 6 animals after 7 to 10 days. Two animals showed persistent B cell depletion until study termination, with two others showing a temporary response. The presence of vector copy could be detected in the peripheral blood of all treated animals between days 3 and 10, and in isolated spleen cells in 5 of 6 animals. All control animals (saline) were negative for vector. RT-ddPCR mRNA expression similarly revealed the presence of anti-CD20CAR transcripts in isolated spleen cells from treated animals; no expression was detected in tissues from control animals. Elevations in inflammatory cytokines could be detected in the serum of treated animals between days 3 and 14. Fusosome treatment was well-tolerated in all animals with no evidence of adverse effects. Moreover, T cell transduction and B cell depletion was not associated with cytokine-related toxicities, and blood chemistry and histopathology were within normal limits. Conclusion: These data obtained in an immunologically competent animal demonstrate the proof-of-concept that systemic administration of a CD8a-anti-CD20CAR fusosome can specifically transduce T cells in vivo without pre-conditioning or T cell activation, resulting in B cell depletion in the absence of vector- or CAR T-related toxicities. Therefore, fusosome technology represents a novel therapeutic opportunity to treat patients with B cell malignancies and potentially overcome some of the treatment barriers that exist with conventional CAR T therapies. Disclosures Cunningham: Sana Biotechnology: Current Employment. Chandra: Sana Biotechnology: Current Employment. Emmanuel: Sana Biotechnology: Current Employment. Mazzarelli: Sana Biotechnology: Current Employment. Passaro: Sana Biotechnology: Current Employment. Baldwin: Sana Biotechnology: Current Employment. Nguyen-McCarty: Sana Biotechnology: Current Employment. Rocca: Sana Biotechnology: Current Employment. Joyce: Sana Biotechnology: Current Employment. Kim: Sana Biotechnology: Current Employment. Vagin: Sana Biotechnology: Current Employment. Kaczmarek: Sana Biotechnology: Current Employment. Chavan: Sana Biotechnology: Current Employment. Jewell: Sana Biotechnology: Current Employment. Lipsitz: Sana Biotechnology: Current Employment. Shamashkin: Sana Biotechnology: Current Employment. Hlavaty: Sana Biotechnology: Current Employment. Rodriguez: Sana Biotechnology: Current Employment. Co: Sana Biotechnology: Current Employment. Cruite: Sana Biotechnology: Current Employment. Ennajdaoui: Sana Biotechnology: Current Employment. Duback: Sana Biotechnology: Current Employment. Elman: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Harding: Sana Biotechnology: Current Employment. Lyubinetsky: Sana Biotechnology: Current Employment. Patel: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Ruzo: Sana Biotechnology: Current Employment. Iovino: Sana Biotechnology: Current Employment. Varghese: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment. Gorovits: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. McGill: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dambach: Sana Biotechnology: Current Employment.

CD4-Targeted Fusosomes Are Capable of Transducing Resting T Helper Cells to Generate Highly Potent CAR-T Cells

Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells. Methods: Anti-CD4 single chain variable fragments () and single variable domain (VHHs) were screened for CD4 binding, specificity, and NHP cross-reactivity and inserted into receptor binding paramyxovirus fusogen. CD4-targeted fusosomes expressing GFP were screened for high on-target titer against the CD4+ SupT1 cell line and low off-target transduction on non-CD4 expressing cells. Subsequently, a CD19-specific CAR encoding 4-1BB and the CD3z endo-domains (CD19 CAR) was generated to examine CD4+ CAR T transduction efficiency and functionality. PBMCs were thawed and activated with anti-CD3/anti-CD28 beads and exposed to GFP, CD4-targeted fusosomes and specificity of targeting CD4+ T cells was measured by flow cytometry. Subsequently, CD19 CAR fusosomes targeting CD4 were used to test transduction efficiency against activated (CD3/CD28 or IL-7 treated) or resting T cells, and to measure T cell function against CD19+ and CD19 knockout (CRISPR/Cas9-edited) Nalm-6 lymphoma cells (e.g., tumor co-culture and rechallenge assays and cytokine production) in vitro. Vector copy number (VCN) was determined by a multiplex ddPCR assay and reported as copies per diploid genome (c/dg). Results: To target CD4+ T cells, we generated fusogens encoding scFvs and VHHs specific to the CD4 T cell co-receptor (n = 399). Using fusosomes carrying the GFP transgene, NHP cross-reactive CD4-targeted fusogens that efficiently transduced CD4+ SupT1 cells were selected (n = 12 with crude SupT1 titers >1E6). Activated PBMCs transduced with a CD4-targeted fusosomes exhibited specific CD4 T cell transduction whereas VSV-G pseudotyped vectors showed broad transduction including CD4+ and CD8+ T cells. CD4-targeted CD19 CAR fusosomes could efficiently transduce both activated (34% ± 1.5% CD4+CAR+; 0.54 ± 0.18 c/dg) and resting T cells, albeit at a lower expression and integration rate (20% ± 0.5% CD4+CAR+; 0.28 ± 0.14 c/dg). Resting CD4-transduced CAR T cells demonstrated specific cytotoxicity and cytokine production (GM-CSF, IFN-g, TNF-a, IL-2, IL-6, and IL-10) against CD19+ Nalm-6 but did not recognize CD19 knockout tumor cells. In long-term co-culture assays with repetitive stimulation with fresh tumor cells, resting CD4+ CD19 CAR T cells continued to show potent tumor cell killing. Future experiments will evaluate the efficacy of CD4 fusosomes against CD19+ tumors in vivo. Summary: CD4-specific fusosomes can efficiently deliver an integrating CAR payload to resting and activated CD4+ T cells. Modified CD4+ CAR T cells demonstrate potent anti-tumor activity against CD19+ tumor cells. These data suggest that targeting the CD4 co-receptor through in vivo delivery using a novel pseudotyped integrating vector can produce functional CAR T cells to target cancer. Disclosures Ciarlo: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. DeGroot: Sana Biotechnology: Current Employment. Flores: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Johnson: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

IMPACT OF EXPORTS AND IMPORTS ON THE ECONOMIC GROWTH IN BURUNDI

This paper investigated the impact of exports and imports on the economic growth in Burundi. To achieve this purpose, annual data for the periods between 1989 and 2018 were tested. The study used Granger Causality and Johansen Co-integration approach for long-run relationship Using Augmented Dickey-Fuller (ADF) and Phillip-Perron (PP) stationarity test, the variable proved to be integrated of the order I(1) at first difference. Johansen and Juselius Co-integration test was used to determine the presence or otherwise of a co-integrating vector in the variables. To find out the direction of causality among the variables, at least in the short-run, the Pairwise Granger Causality was carried out. Exports were found to Granger Cause imports. The results show that there is unidirectional causality between exports and imports. These results provide evidence that growth in Burundi was propelled by a growth-led import strategy as well as export-led import. Imports are thus seen as the source of economic growth in Burundi. KEYWORDS: Co-integration, Granger causality; Exports; Imports; Economic growth, Burundi.

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Dynamic Inflation and Economic Growth Nexus in Bangladesh

This scholarly article seeks to spotlight the inextricable link between economic expansion and inflation in Bangladesh for the past three decades from 1987 to 2017. The nature of the relationship between these two macroeconomic variables is a boiling topic of research. The data on both the GDP growth and inflation rates supplied by the World Bank have been used to study the nexus. Different relevant tests (DF, ADF, PP and KPSS test) found unit root in the variables, but this problem is disappeared at the first difference. Cointegration tests display the long-run connection between the variables at the period. Max-Eigen value Statistic Trace Statistic expose there may be a second integrating vector. The vector error correction model (VECM) finds short dynamics among inflation and economic development, and the adjustment speed at 39% and 82% respectively for the variables—GDP growth rate and inflation. This empirical study has found a significant correlation between inflation and economic growth in Bangladesh during the study period

The Nexus Between Economic Globalization And Female Labor Force Participation

Female labor participation is now being considered as one of the main elements of development among policy makers, feminist, sociologists and economists. Female labor supply is an essential factor not only for economic development but also for socio-economic development of a nation. This paper empirically examines the nexus between economic globalization and female labor force participation (FLFP) for Pakistan from 1973 to 2014. Furthermore, this study also explores the role of economic stability through female’s unemployment rate, inflation rate and per capita income, and increase in productivity through human capital investment on female labor force. This study also finds out the impact of gender disparity on female labour force participation in Pakistan. For estimation of result, Autoregressive Distributive Lag Model ARDL approach to co-integration has been applied which identifies co- integrating vector(s). After identification of co-integrating vector(s) ARDL model is re-parameterized into ECM. The empirical finding proves a positive and significant relationship between economic globalization and FLFP in long run as well as in short run for Pakistan. Beyond labor laws, policy maker should focus on whether female will get new job opportunities and benefits of new markets especially for females when a country opens it economy. For this purpose, it is necessary to improve access to education for females and developing their skills by providing training program, promote children care centers, legal measures to reduce the burden of domestic chores, and encourage private sector development in different sectors especially in industries that may help to increase employment opportunities for females in Pakistan.

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

ABSTRACTCaldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in thepyrFgenome region. ThepyrFgene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand theC. besciigenetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs inC. bescii. These strains showed resistance to kanamycin at concentrations >50 μg · ml−1, whereas wild-typeC. besciiwas sensitive to kanamycin at 10 μg · ml−1. In addition, placement of the Cbhtkmarker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of thepyrEgene in wild-typeC. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. ThepyrEgene functioned as a counterselectable marker, likepyrF, and was used together with Cbhtkin the ΔpyrEbackground strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme.IMPORTANCECaldicellulosiruptor besciiis a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used withC. besciiis based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system forC. besciiusing the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.