Kim, Sarah2022-04-132022-04-132021-12https://hdl.handle.net/11299/226951University of Minnesota Ph.D. dissertation. December 2021. Major: Experimental & Clinical Pharmacology. Advisors: Jeanine Jarnes, James Cloyd. 1 computer file (PDF); xiv, 227 pages.Lysosomal diseases are a group of over 70 diseases with a combined incidence of approximately 1 in 7,700 live births. Most lysosomal diseases are caused by mutations in enzymes normally present in the lysosome. Lysosomal diseases are multi-systemic and progressive diseases. Currently, only 12 lysosomal diseases have treatments. A challenge in drug development is the lack of biomarkers that reflect disease progression or show response to therapy. Furthermore, current therapies have difficulty reaching certain tissues that are major contributors to morbidity and mortality, such as the central nervous system (CNS) and cardiac valves. The overall objective of this dissertation is to improve the delivery of therapeutics to targeted tissues in lysosomal diseases. To accomplish this objective, three main studies were performed. 1) Validation of Chitotriosidase as a CNS Biomarker for Gangliosidoses. Chitotriosidase was investigated as a probable surrogate endpoint for clinical trials with gene therapy. The first objective was to validate chitotriosidase levels for important clinical outcomes in patients with lysosomal diseases. The second objective was to assess chitotriosidase’s ability to detect effective gene therapy in murine models of lysosomal diseases. In patients with gangliosidoses, the most severe infantile phenotype had higher chitotriosidase levels in the cerebrospinal fluid (CSF) and a different pattern over time than the more attenuated juvenile and late-onset forms. Chitotriosidase levels were also significantly associated with neurocognitive impairment. In mice with mucopolysaccharidosis type I (MPS I), there were significant differences among the untreated, gene-therapy treated, and mice heterozygous for a mutation in the IDUA gene. These results support the use of CSF chitotriosidase levels to diagnose different disease phenotypes and to monitor disease progression in patients. As a potential biomarker of neurological improvement, CSF chitotriosidase can aid in the development of therapies that target the CNS. 2) Investigation of Iduronidase Enzymes Linked to Pepcan to Improve Delivery to Targeted Tissues. The objective of this study was to determine if pepcan-12 can increase the uptake of iduronidase into the brain of MPS I mice. Pepcan-12 is a ligand for the cannabinoid receptor type 1 (CB1), a highly expressed receptor in the CNS. The hypothesis was that a fusion iduronidase containing pepcan-12 would have higher activity levels than iduronidase in the brain. Sequences of one of two linkers, Linker S or Linker T, were inserted between pepcan-12 and IDUA to conjugate the ligand and iduronidase. MPS I mice were injected with plasmids encoding either the native iduronidase or one of four fusion iduronidase enzymes containing: pepcan-12 + Linker S, pepcan-12 + Linker T, Linker S, or Linker T. The fusion enzymes and iduronidase had similar activity levels in the brain. Unexpectedly, the fusion enzymes had higher activity levels than iduronidase in the heart and plasma, which appears to be caused by the linkers. Therefore, these fusion enzymes may improve cardiovascular outcomes in MPS I. In several MPS disorders, the cardiac valves continue to worsen despite enzyme replacement therapies (ERT) and hematopoietic cell transplants. The small size of these linkers facilitates their use as fusion enzymes encoded in gene therapy or administered directly as ERT. Therefore, these linkers may aid in therapeutic development for other lysosomal diseases. 3) Pharmacokinetic Analysis of Iduronidase and a Fusion Iduronidase Enzyme Encoded in Gene Therapy. In the previous study, an iduronidase enzyme containing Linker T, termed Linker T iduronidase, had higher activity levels than iduronidase in the plasma and heart. This study sought to investigate the mechanism of Linker T iduronidase, but a gap between the fields of lysosomal diseases/gene therapy and pharmacokinetics (PK)/ pharmacodynamics (PD) became apparent. In the field of lysosomal diseases, the activity level of an enzyme is an important measurement of efficacy, because an enzyme’s activity levels are more predictive of efficacy than its physical levels. However, pre-clinical studies in lysosomal diseases lack well-described methods to quantify changes in enzyme activity levels over time. In contrast, the pharmacokinetic field has rigorous and reproducible methods to quantify changes in a therapy over time in the body. However, traditional pharmacokinetic methods face challenges in gene therapy because of the need for uniform or convertible units. Furthermore, absorption, distribution, metabolism, and elimination processes are well-characterized for small molecule drugs but not yet adapted for biological therapies. To bridge the fields of lysosomal diseases/gene therapy and PK/PD, I aimed to develop an approach incorporating values with greater prediction of efficacy from the field of lysosomal diseases and the quantitative methods from the field of pharmacokinetics. The objective of this study was to perform a pharmacokinetic analysis of iduronidase and Linker T iduronidase administered as gene therapies. The hypothesis was that Linker T iduronidase would have a higher area under the curve (AUC) or half-life, estimated with enzyme activity levels, than iduronidase in the plasma. MPS I mice were injected with plasmids encoding either iduronidase or Linker T iduronidase. At ten time points, ranging from 0.5 to 168 hours post-injection, the enzymes’ physical levels in the liver, activity levels in the liver, and activity levels in the plasma were measured. In the liver, both the physical and activity levels over time were similar between the native iduronidase and Linker T iduronidase. In contrast, enzyme activity levels over time in the plasma showed differences between the native iduronidase and Linker T iduronidase. The time curves of activity in the plasma showed biphasic profiles for both enzymes. Iduronidase had a sharper decline between 24 and 48 hours, and both enzymes had approximately parallel slopes between 96 and 168 hours. The Linker T iduronidase had a two-fold higher AUC of activity than the normal iduronidase in the plasma. The AUC of plasma activity and other PK parameters were contextualized in gene therapy, and experimental data were used to deduce the mechanism of Linker T. These results suggest that Linker T iduronidase may have a distinct property that protects the enzyme from degradation or inactivation in the plasma. The enzymes were estimated to have a half-life of activity in the plasma under noncompartmental analysis. Future studies with compartmental analysis would better characterize half-lives of activity in biphasic profiles. This study performs a novel approach of conducting a formal pharmacokinetics analysis on enzyme activity levels, a traditionally pharmacodynamic outcome. The resulting PK parameters can be interpreted and used to gain mechanistic insight on gene therapy, by integrating concepts from pharmacokinetics and gene therapy In summary, these findings improve the therapeutic delivery in lysosomal disease through the validation of a CNS biomarker for lysosomal diseases, creation of a fusion enzyme with improved activity in the heart and plasma, and a novel approach and interpretation of pharmacokinetics to gain mechanistic insight on gene therapy.enBiomarkerEnzyme replacement therapyFusion enzymeGene therapyLysosomal diseasesPharmacokineticsThesis or Dissertation