Browsing by Subject "Omics"

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    Genomic Analysis And Engineering Of Chinese Hamster Ovary Cells For Improved Therapeutic Protein Production
    (2020-05) O'Brien, Sofie
    Protein biologics have transformed the field of medicine in recent years. These complex molecules are produced in living cells, primarily Chinese Hamster Ovary (CHO) cells. Due to the importance of these therapeutic proteins to disease treatment, it is essential to improve the efficiency of their production, both to promote the development of new therapies, and to bring down the cost of manufacturing. One of the most important components of the production process is the development of a cell line. Many features of a cell line, such as cellular growth, metabolism, and the integration site of the gene encoding the protein, influence the resulting culture productivity and quality of the protein produced. In this thesis, multiple aspects of the relationship between integration site and resulting cell line behavior were investigated. First, a rapid integration site identification method was developed to facilitate further analysis of integration sites in complex cell lines. Next, to examine genomic instability, parental cells were compared with high and low producing subclones, leading to identification of genomic regions vulnerable to copy gain/loss. A large-scale analysis across many CHO cell lines was further performed to look for global regions of genomic variation, independent of an individual cell line. To evaluate integration sites with high transcriptional potential, integration sites from high producing cells were examined, and high transgene expression was correlated to high transcriptional activity and accessibility of the integration region. This work also extended to energy metabolism, another key feature of a cell line. Through the use of model guided multi-gene engineering to manipulate cell metabolism, waste product generation was reduced in late-stage culture. With these tools and technologies, we can build a more complete picture of a desirable integration site, which can be used to drive the development of next generation cell lines with high, stable expression of transgenes for therapeutic protein production.
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    Using Genetics to Understand and Overcome CART Cell Resistance and Toxicities
    (2022-04) Cox, Michelle
    Chimeric antigen receptor T (CART) cell therapy is an engineered cellular therapy that redirects T cells to cancer cells expressing certain antigens. CD19-directed CART cell therapy is the most advanced CART cell therapy in the clinic and is currently approved for the treatment of different B cell malignancies. However, the wider application of CART cell therapy in hematological malignancies is limited by its toxicities and lower rates of durable remission. Through translational and correlative science, large data analysis, bioinformatics, and advances in synthetic biology, we have learned that a predominant mechanism of CART cell therapy resistance is the immunosuppressive tumor microenvironment (TME). The main objective of this work was to understand how the TME impacts CART cell functions and to create inhibition-resistant CART cells utilizing genetic sequencing and synthetic biology tools. Specifically, we aimed to investigate the mechanisms by which 1) granulocyte-macrophage colony-stimulating factor (GM-CSF) and 2) leukemic extracellular vesicles (EVs) impact CART cell functions.In the first part of this work, we have discovered that GM-CSF directly impacts CART cells through modulation of their activation pathways. We have previously demonstrated that GM-CSF contributes to myeloid cell activation and to the development of toxicities after CART cell therapy. Using CRISPR/Cas9, we generated GM-CSFKO CART19 cells and demonstrated their reduced production of GM-CSF. GM-CSFKO CART19 cells demonstrated enhanced proliferation and superior anti-tumor activity in preclinical models, suggesting a direct effect of GM-CSF disruption on CART cells, independent of its known modulation of myeloid cells. To investigate the mechanism of this enhanced efficacy, we first ruled out off-target editing by performing whole exome sequencing. We then interrogated the transcriptome of GM-CSFKO CART19 cells, which showed a distinct gene expression profile suggesting alteration of activation pathways. We validated this immunophenotype on a protein level and its effect on CART cell activation and functions in vivo. In the second part of this work, we discovered a novel mechanism of resistance to CART cell therapy through their inhibition by tumor-derived extracellular vesicles. In this work, we used chronic lymphocytic leukemia (CLL) as a model to interrogate these interactions. The immunosuppressive microenvironment in CLL is well known to inhibit effector immune cells and in part may be related to the abundance of circulating EVs bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. To test this hypothesis, we first enumerated and immunophenotyped circulating EVs from platelet-free plasma in untreated patients with CLL. We determined their interaction with CART19 cells and found that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation and killing. Our mechanistic studies demonstrated that CLL-derived EVs induce a state of T cell dysfunction characterized by functional, immunophenotypical, and transcriptional hallmarks of exhaustion and this dysfunction is more specific for PD-L1high EVs. In conclusion, we demonstrate that 1) CRISPR/Cas9 GM-CSF knockout in CART cells modulates their activation and enhances overall expansion and 2) leukemic EVs induce significant CART19 cell dysfunction by altering exhaustion pathways. GM-CSFKO CART19 is a novel CART cell therapy that is potentially less toxic and more effective than current CART19. The knowledge that leukemic EVs induce CART cell dysfunction paves the way for future studies of EV phenotype and cargo, which can ultimately lead to new strategies to predict outcomes and implement individualized CART cell therapy.