Versatile Applications of Stem Cell Gene Therapy: From Vector Biology to Immunological and Neurological Disease Correction

This dissertation presents the development and application of gene therapy and genome editing strategies for the treatment of rare genetic diseases using hematopoietic stem cells (HSCs) as vehicles for durable therapeutic benefit. Chapter 1 characterizes recombinant adeno-associated virus (rAAV) vector heterogeneity, highlighting the prevalence of DNA contaminants in rAAV preparations and their detrimental effects on hematopoietic stem and progenitor cell (HSPC) function, clonogenicity, and genome editing outcomes. Utilizing Single-Stranded Virus sequencing (SSV-seq), we identify variability in DNA impurities across preps and demonstrate their impact on editing efficiency and transcriptional profiles in CD34+ HSPCs. Chapter 2 develops a site-specific genome editing approach for X-linked agammaglobulinemia (XLA) by inserting a BTK transgene into its endogenous locus using CRISPR-Cas9/rAAV6. Edited murine Lin– cells engrafted into XLA mice resulted in reconstitution of B cells, immunoglobulin production, and improved humoral immunity, validating the therapeutic potential of endogenous BTK correction. Chapter 3 advances a cross-corrective lentiviral gene therapy for Angelman syndrome (AS) by engineering a lentiviral vector expressing a secreted form of UBE3A driven by safer, codon-optimized promoters. In a mouse model of AS, transplantation of transduced HSCs restored UBE3A in neurons and improved motor, cognitive, and electrophysiological phenotypes to wildtype levels. Together, this work advances novel applications of HSC gene therapy and editing platforms to correct inherited immune and neurodevelopmental disorders and addresses critical vector design and manufacturing challenges that influence therapeutic efficacy

DNA contamination within recombinant adeno-associated virus preparations correlates with decreased CD34+ cell clonogenic potential.

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPCs), but they have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in the packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage single-stranded virus (SSV) sequencing, a next-generation sequencing-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34+ HSPCs with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential

Cutting Edge: Hypoxia Sensing by the Histone Demethylase UTX (KDM6A) Limits Colitogenic CD4+ T Cells in Mucosal Inflammation.

Hypoxia is a hallmark of inflammatory conditions (e.g., inflammatory bowel disease [IBD]), and adaptive responses have consequently evolved to protect against hypoxia-associated tissue injury. Because augmenting hypoxia-induced protective responses is a promising therapeutic approach for IBD, a more complete understanding of these pathways is needed. Recent work has demonstrated that the histone demethylase UTX is oxygen-sensitive, but its role in IBD is unclear. In this study, we show that hypoxia-induced deactivation of UTX downregulates T cell responses in mucosal inflammation. Hypoxia results in decreased T cell proinflammatory cytokine production and increased immunosuppressive regulatory T cells, and these findings are recapitulated by UTX deficiency. Hypoxia leads to T cell accumulation of H3K27me3 histone modifications, suggesting that hypoxia impairs UTXs histone demethylase activity to dampen T cell colitogenic activity. Finally, T cell-specific UTX deletion ameliorates colonic inflammation in an IBD mouse model, implicating UTXs oxygen-sensitive demethylase activity in counteracting hypoxic inflammation

A millennial record of environmental change in peat deposits from the Misten bog (East Belgium).

In this study, palaeoenvironmental changes recorded in the top metre of a peat profile (Misten bog, East Belgium) were investigated using a multiproxy approach. Proxies include bulk density, Ti and Si content, pollen, macrofossils, d13C on specific Sphagnum stems, and d13Ced18O on Sphagnum leaves. A highresolution chronology was generated using 210Pb measurements and 22 14C AMS dates on carefully selected Sphagnum macrofossils. d13C only records large change in mire surface wetness. This is partly due to the fact that the core was taken from the edge of a hummock, which may make it difficult to track small isotopic changes. The d13C signal seems to be dependent upon the Sphagnum species composition. For example, a change between Sphagnum section Cuspidata towards Sphagnum imbricatum causes a significant drop in the d13C values. On the whole, the C and O isotopes record two shallow pool phases during the 8the9th and the 13th centuries. Pollen and atmospheric soil dust (ASD) fluxes records increased human occupation in the area. There may be some climatic signals in the ASD flux, but they are difficult to decipher from the increasing human impact (land clearance, agriculture) during the last millennium. The variations in the proxies are not always synchronous, suggesting different triggering factors (temperature, wetness, windiness) for each proxy. This study also emphasizes that, compared to studies dealing with pollution using geochemical proxies, palaeoclimatic inferences from peat bogs need as many proxies as possible, together with highly accurate and precise age-models, in order to better understand climate variability and their consequences during the Holocene

PROPEMPTICA || IN DISCESSVM || REVERENDI ET ORNA=||tiß: viri, eruditione et pietate praestantis, Do-||mini M. DAVIDIS BRAMERI Brun-||suicensis, Diaconi Ecclesiae VVitebergensis,|| vocati ad officium Pastoris in || oppido Pesenick.|| Scripta ab Amicis.||

Vorlageform des Erscheinungsvermerks: VVITEBERGAE || Excudebant Clemens Schleich et Anto=||nius Schöne.|| ANNO M.D.LXXIII.|