Date of Award
5-14-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
In recent years, engineered cells have been successfully developed for a variety of therapies including cancer and genetic disorders using cells such as T lymphocytes and hematopoietic stem cells. A novel approach that has been enabled by the advent of CRISPR/Cas gene editors is the engineering of B lymphocytes. B lymphocytes are the antibody producing cells of the adaptive immune system and are pivotal in control of pathogens through the binding of membrane bound antibody, or the B cell receptor, to cognate antigen. In conjunction with appropriate co-stimulatory signals, this binding results in activation of the cells and eventual secretion of soluble antibody into the bloodstream. The unique features of B lymphocytes have made them an attractive target for engineering, with several studies pursuing their use for control of infectious diseases. This is achieved through insertion of novel broadly neutralizing antibodies into the endogenous B cell receptor genetic locus using CRISPR/Cas gene editors. While promising, such studies almost exclusively utilize ex vivo cell editing, a lengthy and complex process requiring removing the patients cells, engineering them, and infusing them back to the patient. A broad vaccination campaign to achieve control of infectious disease will likely require new technologies beyond ex vivo engineering. An alternative is direct in vivo engineering, whereby a vector encoding the necessary machinery for gene editing is delivered to the patient and directed towards the cells of interest. This could circumvent many of the challenges and costs associated with ex vivo cell engineering and enable the delivery of novel medicines to broad patient populations. Towards this end we pursued the development of Adenoviral vectors (Ads) for in vivo B lymphocyte gene transfer. Ads have recently been produced at low cost and global scales to control the SARS-CoV-2 pandemic. Successful development of an Ad for in vivo B lymphocyte engineering could thus dramatically expand the scope of B lymphocyte-based therapies beyond the capacity of current technologies. In this work we first established that Ads were capable of gene expression in ~1% of B lymphocytes in vivo after systemic delivery. We established an unexpected mechanism for this gene transfer, and found we could modify these Ad vectors to drive gene expression from a B lymophocyte-specific promoter. One exemplary promoter completely restricted gene expression to B lymphocytes both in vitro and in vivo, resulting in a multi-log enhancement in the targeting ratio. However, the use of such promoters reduced the overall level of gene expression, potentially limiting the utility of our vector. We thus pursued the development of novel Ads functionalized with molecular glue peptides to achieve decoration of the vector capsid with antibodies targeting receptors on B lymphocytes. Through this technology we were able to demonstrate dramatic gene transfer augmentation in vitro and a roughly 2-fold enhancement in vivo.
Language
English (en)
Chair
David Curiel