Engineered Platforms for Gene and Cell Therapies

The fields of gene and cell therapy have transformed biomedical research by providing a path towards personalized medicine. The advent of whole genome sequencing and high throughput screening technologies has generated a wealth of information on understanding the causes and effects of many diseases. In addition, two technologies in particular, human pluripotent stem cells and CRISPR-Cas9, are redefining the fields by capitalizing on disease pathology data and providing methods to target the underlying cause of a disease. The success of these technologies for clinical applications, however, depends on the development of safe, scalable, and efficient methods for translation into the human body. In this dissertation, two engineered platforms are introduced to address several challenges associated with scalable production and long term safety of human pluripotent stem cells and CRISPR-Cas9, respectively./nHuman pluripotent stem cells offer considerable potential for biomedical applications including drug screening and cell replacement therapies. Clinical translation of human pluripotent stem cells requires large quantities of high quality cells, so scalable methods for cell culture are needed. However, current methods are limited by scalability, the use of animal-derived components, and low expansion rates. Here, a scalable 3D biomaterial with the capacity to tune both the chemical and mechanical properties is demonstrated to promote human pluripotent stem cell expansion under defined conditions. This 3D biomaterial, comprised of hyalauronic acid and poly(N-isopropolyacrylamide), has thermoresponsive properties that readily enable mixing with cells at low temperatures, physical encapsulation within the hydrogel upon elevation at 37 ºC, and cell recovery upon cooling and re-liquefaction. After optimization, the resulting biomaterial supports human pluripotent stem cell expansion over long cell culture periods while maintaining cell pluripotency. The capacity to modulate the mechanical and chemical properties of the hydrogel provides a new avenue to expand hPSCs for future therapeutic application./nCRISPR-Cas9 allows for precise genomic modifications with relative ease of design. Adeno-associated virus is a leading candidate delivery vehicle for CRISPR-Cas9 because of its broad clinical potential and ability to transduce a broad range of cell types, including non-dividing cells. However, a major limitation to this approach is the formation of stable episomal DNA that leads to indefinite Cas9 expression in the cell. Persistent Cas9 expression can lead to potential off-target editing and immune responses to the bacterial Cas protein. Here, several engineered strategies to inactivate Cas9 activity are compared to identify the design with optimal kinetics for in vivo therapeutic editing. A dual sgRNA approach where a Cas9 targeted sgRNA is co-delivered with a gene targeting sgRNA provided the most potent levels of self-inactivation. Furthermore, tunable control of Cas9 inactivation was shown through incorporation of mismatches in the Cas9 targeted sgRNA. The resulting self-inactivation system retained therapeutic genome editing in vivo. A tunable self-inactivation strategy for adeno-associated virus mediated delivery of CRISPR-Cas9 provides a safer approach for therapeutic genome editing.

Advisor: Schaffer, David V