Synthetic Peptide Analogues as Targeting and Endosomal Guiding Reagents for Gene Therapy of Autoimmune Diseases

Autoimmune diseases are a broad group of diseases, all caused by inappropriate immune responses against self-antigens, resulting in tissue damage and chronic disease. Conventional therapies focus on symptom management and broad immunosuppression, which lack specificity and come with risks such as infection and malignancy. Novel strategies, such as immunomodulating peptides and small interfering RNA (siRNA), have emerged as potential approaches to selectively target pathogenic immune cells while preserving normal immune function. However, key challenges remain, including the identification of disease-relevant epitopes and the development of delivery systems that ensure efficient cellular uptake and endosomal release. This thesis addresses some of these challenges.

Epitope mapping is conducted for autoantigens relevant to type 1 diabetes and Graves’ disease. In type 1 diabetes, a preproinsulin-derived peptide (preproinsulin_4–13) is identified as a B-cell epitope. The peptide binds to antibodies in the serum of type 1 diabetes patients and interacts with patient-specific B-cells. This epitope overlaps with previously reported T-cell epitopes, and residues critical for T-cell recognition are also shown to be essential for antibody binding, suggesting sharing of epitopes between lymphocytes and potential B–T cell crosstalk. In GD, potential epitopes within the LRR and hinge regions of the thyroid-stimulating hormone receptor are identified through a combination of computational and experimental epitope mapping. None of these linear peptides, however, shows consistent disease-specific reactivity across larger patient cohorts, reflecting both the heterogeneity of immune responses in Graves’ disease patients and the likely importance of conformational, rather than linear, epitopes. The therapeutic potential of a preproinsulin_4–13 is, evaluated in vivo in murine models of diabetes. The peptide, formulated with LPX, demonstrates dose-dependent improvements in glucose metabolism and insulin sensitivity in diet-induced models, whereas a scrambled peptide control shows minimal effect. The observed effects, however, are transient and vary between strains, and no clear benefit is observed in NOD mice during the first weeks after administration. Extended monitoring and additional analyses, including histology, immune cell profiling, and RNA sequencing, will determine whether delayed or subtle immunological effects emerge. Additionally, siRNA targeting TNFRSF13C (BAFFR), an important factor for B-cell survival and proliferation, has been developed and evaluated in human and murine B-cell lines, demonstrating efficient gene knockdown. Conjugates between TNFRSF13C siRNA and preproinsulin_4–13 peptide analogues are synthesized and tested. Conjugation to a linear peptide is shown to preserve knockdown efficiency, whereas bulkier, branched peptide conjugates partially reduce activity. These results suggest that disease-relevant peptides can be conjugated to siRNA, providing the potential to target pathogenic B-cells, while preserving functional gene silencing.

Finally, strategies for enhancing intracellular delivery are explored using pH-sensitive masking of cell-penetrating peptides with maleic anhydride derivatives. Cyclohexene-1,2-dicarboxylic anhydride shows favorable pH-dependent masking and de-masking, efficiently protecting CPPs at physiological pH and restoring activity under acidic conditions. Masking efficiency and de-masking kinetics vary with peptide sequence, demonstrating the need to tailor masking strategies to specific CPPs.