Integration site-based clonality analyses of potency of stem and progenitor cells and safety in human gene and cell therapy

Since their development over thirty years ago, replication incompetent retroviral vectors have become indispensable tools for stable genetic modification in gene therapy and experimental biology (Mann et al. 1983). One notable feature of these vectors is that they irreversibly integrate their genome into the genomic DNA of the target cell, where it is copied along with the cellular genes and passed on to daughter cells when the cell proliferates. Because integration occurs at a random location, each transduced cell within a population of reasonable complexity acquires a retroviral genome at a unique site. The integration process does exhibit some preferences, but the process is still random and most genomic sites are amenable to integration. By using methods that can distinguish between these distinct integration sites (ISs) based on their unique properties, one can enumerate the ISs present in a cellular population. More interestingly, these unique properties can be observed over time as cellular clones expand, contract, differentiate, migrate, die or express other behaviors, allowing one to make inferences about clonal biological potential and proliferative dynamics.This concept, which is most commonly referred to as “clonal tracking,” will occupy the entirety of this thesis aside from chapter 2. Chapter 2 presents two works improving the function and production of lentiviral vectors based on human immunodeficiency virus 1 (HIV-1), a slightly newer retroviral genetic engineering technology that has almost completely superseded that of retroviral vectors based on murine viruses (Cooper et al. 2011; Cooper et al. 2015). Chapter 3 contains two studies in which clonal tracking was used to demonstrate the ability of either hematopoietic or cancer stem cells to produce multiple distinct cell types (Stoyanova et al. 2013; McCracken et al. 2013). Chapter 4 describes experiments in which clonal tracking was used to determine whether a recently discovered immunophenotypically defined population of pluripotent stem cell-derived progenitor cells possesses multipotency (Chin, Cooper et al., Stem Cells, In Press). The unprecedented complexity of ISs sequenced in this work prompted a more thorough statistical examination of the issue of sampling in IS sequencing approaches. Chapter 5 contains multiple published collaborative studies in which IS sequencing was used to examine the safety of gene and cell engineering approaches for therapeutic applications in gene therapy or regenerative medicine (Karumbayaram et al. 2012; Awe et al. 2013; Candotti et al. 2012; Carbonaro et al. 2014; Romero et al. 2013; Hoban et al. 2015). Chapter 6 discusses work on the safety and efficacy of retroviral gene therapy in a human clinical trial for adenosine deaminase- deficient severe combined immunodeficiency (ADA-SCID). In addition to applying the molecular and statistical methods for IS-based clonal tracking developed during my graduate work, this analysis also involved large-scale sequencing of the T-cell receptor rearrangements that are the foundation of the adaptive immunological repertoire. Chapter 7 summarizes the work and discusses unanswered questions for potential future research.