Biology subjects

Ballek, O.

Publications and source records attributed to Ballek, O..

3 recordsLinked to original sources

bioRxiv2026

Adeno-Associated Virus Co-Precipitation with Extracellular Vesicles for Genome Editing in Rodent Embryo

Adeno-associated virus purification by density-gradient ultracentrifugation is labor-intensive and often results in substantial titer loss due to particle aggregation. Here, we present a scalable co-isolation strategy in which AAV is precipitated together with extracellular vesicles secreted by the producer cell line, completely bypassing density-gradient separation. The resulting AAV-EV preparations comprise free AAV, free EVs, and EV-associated AAV. Functionally, AAV-EV vectors (AAV2/1 serotype) support efficient ex vivo genome editing across multiple independent loci in mouse and rat zygotes, achieving a mean targeting efficiency of approximately 26%. Compared with gradient-purified AAV administered at matched doses, AAV-EV formulations yielded 2.34-fold higher embryo viability while maintaining equivalent transgene copy numbers. By leveraging EVs as a biological matrix, this approach enables ultracentrifugation-free AAV isolation without compromising vector functionality. Overall, AAV-EV represents an accessible and embryo-tolerant platform for rodent genome engineering that aligns with the principles of Replacement, Reduction, and Refinement (3R) principles.

bioengineering↗

bioRxiv2025

Claudin 1-mediated positioning of DC1 to mTECs is essential for antigen transfer-coupled DC1 maturation and maintenance of central tolerance

The mechanisms of central tolerance, which rely on the presentation of self-antigens by medullary thymic epithelial cells (mTECs) and DCs, prevent autoimmunity by eliminating self-reactive T-cells. While mTECs produce self-antigens in an autonomous manner, DCs acquire them from mTECs via cooperative antigen transfer (CAT). Our recent data showed that preferential pairing occurs between distinct subsets of mTECs and DCs in CAT, providing a rationale for the existence of molecular determinants which control such pairing and the outcome of central tolerance. Here, we compared the transcriptomes of CAT-experienced and -inexperienced DCs and identified Claudin 1 as a molecule involved in CAT-coupled type 1 DC (DC1) maturation. By mapping thymic DC1 heterogeneity, we identified their early and late maturation states. DC1-specific ablation of Claudin 1 led to a reduction in CAT-experienced late mature DC1s and hampered DC1 maturation. These phenotypes correlated with the displacement of DC1s from the vicinity of mTECs. This translated into impaired Treg selection and clonal deletion of TRA-specific T-cells manifested via a break in tolerance and symptoms of multi-organ autoimmunity. Collectively, our results identify thymic DC1-derived Claudin 1 as a regulator of immune tolerance. One Sentence SummaryThe expression of Claudin 1 on type 1 dendritic cells regulates their proximity to mTECs, which is required for effective antigen transfer coupled with DC1 maturation and establishment of T-cell tolerance.

immunology↗

bioRxiv2021

A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer

Medullary thymic epithelial cells (mTECs) which produce and present self-antigens are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with specific subset of DCs. Using several relevant Cre-based mouse models controlling the expression of fluorescent proteins, we found that in regards to CAT, each subset of thymic DCs preferentially targets distinct mTEC subset(s) and importantly, XCR1+ activated DCs represented the most potent subset in CAT. Interestingly, one thymic DC can acquire antigen repetitively and of these, monocyte-derived DCs (moDC) were determined to be the most efficient in repetitive CAT. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

immunology↗