Biology subjects

Hamann, M. V.

Publications and source records attributed to Hamann, M. V..

4 recordsLinked to original sources

bioRxiv2026

Development of a recombinant adeno-associated virus vector for human T lymphocyte- and natural killer cell-targeted gene therapy

Recombinant adeno-associated virus (rAAV) vectors are widely used for gene delivery but show limited efficiency in immune cells, including T lymphocytes and natural killer (NK) cells. To overcome this barrier, we developed a modular rAAV vector engineering strategy that integrates capsid retargeting with genome optimization. We report a CD7-targeted rAAV vector (CD7-AAV6/9) featuring a nanobody-fused hybrid capsid derived from a rationally selected chimeric combination of AAV6 and AAV9. CD7-AAV6/9 enables efficient and selective transduction of immortalized and primary human T and NK cells in vitro and in vivo in a humanized mouse model, achieves high production titers, and exhibits markedly reduced off-target transduction compared with wild-type serotypes. In parallel, we demonstrate that incorporation of a human gene-derived intron into the vector genome overcomes host-mediated transcriptional repression and enables robust transgene expression in human CD7 T lymphocyte and NK cell populations. To our knowledge, this represents the first application of intron-mediated enhancement in a rAAV vector context. Together, our findings establish an integrated capsid-genome design framework for targeting human T and NK cells, notoriously challenging immune cell populations for gene therapy, and provide a versatile platform readily adaptable to alternative surface markers and therapeutic payloads.

molecular biology↗

bioRxiv2025

Defective proviruses cause T cell reprogramming through promoter exaptation in HIV-1 infection

People living with HIV (PLWH) on antiretroviral therapy (ART) accumulate primarily defective proviral sequences in genomes of often clonally expanded CD4+ HIV-1 target cells. The majority of viral-derived DNA is transcriptionally active and preferentially found at distinct genomic loci suggesting a selective process driven by integration site-specific crosstalk between viral and host sequences. Focusing on one of the most prominent selected integration loci, the BTB Domain and CNC Homolog 2 (BACH2) gene, we here show mechanistic insights how CD4+ T cells are functionally reprogrammed via exaptation of provirus-derived regulatory sequences during long-term ART. Using a cellular model of BACH2-integrated proviruses, we find that proviral transcription drives aberrant BACH2 protein levels that escape autoregulatory feedback and impose BACH2-dependent transcriptomic changes. By mimicking these changes in primary CD4+ T lymphocytes, we observe that BACH2 drives reprogramming of cells toward a proliferative, precursor memory-like type. These reprogrammed CD4+ T cells possess traits of immune evasion and cellular survival that are signatures of persistent HIV reservoir cells in PLWH. Inhibition of provirus transcriptional activity can mitigate exaptation, suggesting a strategy to offset HIV-driven differentiation and expansion of CD4+ T cells. Finally, our data suggest that provirus exaptation at a second prominently selected proviral integration gene, the Signal Transducer And Activator of Transcription 5B (STAT5B) gene, drives a contrary, effector-like T cell fate, suggesting a multifaceted impact of exaptation on immune homeostasis. Overall, our data suggest that transcriptionally active proviruses, even if structurally defective, modulate target cells through insertional activation of integration genes, a process which we postulate to contribute to the complex immune modulation and dysregulation experienced by ART-suppressed PLWH.

microbiology↗

bioRxiv2025

Expression of LTR and LINE1 transposable elements defines atypical teratoid/rhabdoid tumor subtypes

Atypical teratoid rhabdoid tumors (ATRTs) are aggressive central nervous system tumors mainly affecting young children. Extensive molecular characterization based on gene expression and DNA methylation patterns has solidly established three major ATRT subtypes (MYC, SHH and TYR), which show distinct clinical features, setting the basis for more effective, targeted treatment regimens. Transcriptional activity of transposable elements (TEs), like LINE1s and LTRs, is tightly linked with human cancers as a direct consequence of lifting epigenetic repression over TEs. The sole recurrent biallelic loss-of-function mutation in SMARCB1 in ATRTs, a core component of the SWI/SNF chromatin remodeling complex, raises the question of how TE transcription contributes to ATRT development. Here, we comprehensively investigate the transcriptional profiles of 1.9M LINE1 and LTR elements across ATRT subtypes in primary human samples. We find TE transcription profiles are unique, allowing sample stratification into ATRT subtypes. The TE activity signature in ATRT-MYC subtype is unique, setting these tumors apart from SHH and TYR ATRTs. More specifically, ATRT-MYC shows broadly reduced transcript levels of LINE1 and ERVL-MaLR subfamilies. ATRT-MYC is also unique in having significantly less LTR and LINE1 loci with bidirectional promoter activity. Furthermore, we identify 849 differentially transcribed TEs in primary samples, which are predictive towards established ATRT-SHH and-MYC cell line models. In summary, including TE transcription profiles into the molecular characterization of ATRTs might reveal new tumor vulnerabilities leading to novel therapeutic interventions, such as immunotherapy.

cancer biology↗

bioRxiv2025

Spatiotemporal analysis of de novo KSHV infection using Crispr/Cas9-based 3D live cell imaging at single episome resolution

Kaposi Sarcoma-associated herpesvirus (KSHV) persists as a latent episome in infected cells. While the virus efficiently infects established cell lines and primary cells in vitro, the early events guiding establishment of latent infection and the dynamic interplay between viral episomes and host factors remain incompletely understood. Here, we describe the development and application of a CRISPR/Cas9-based 3D live cell imaging system capable of tracking single KSHV episomes in real-time. Our approach exploits the SunTag technology, wherein deactivated Cas9 (dCas9) molecules are fused to repetitive epitope arrays recognized by superfolder GFP-fused single-chain antibodies. By targeting these complexes to terminal repeat units of KSHV, we achieve high level signal amplification, allowing us not only to detect newly incoming viral genomes within the first hours of de novo infection, but also to follow their spatiotemporal trajectories through different stages of the viral lifecycle. Furthermore, to facilitate efficient generation of stable reporter cell lines, we adapted the transposon-based piggyBac system to combine all SunTag components into a single-vector targeting system (SunSeT). Using these systems, we demonstrate the ability to observe both transient and stable interactions between KSHV episomes and key cellular regulators, including the variant polycomb-repressive complex 1 (vPRC) component KDM2B and the innate immune sensor IFI16. Furthermore, our platform allows detailed visualization of episodic changes in episome localization, abundance and distribution during de novo and long-term infection, providing critical insights into how viral genome positioning and dynamics correlate with host subnuclear environments. Overall, our study introduces a robust and adaptable imaging platform to dissect the earliest events of KSHV infection. The ability to track viral episomes in living cells offers a powerful tool to advance our understanding of the spatial and temporal regulation of individual KSHV genomes, shedding light on fundamental mechanisms of herpesvirus latency and persistence.

microbiology↗