Biology subjects

Messana, A.

Publications and source records attributed to Messana, A..

3 recordsLinked to original sources

bioRxiv2023

Comprehensive genome editing confers "off-the-shelf" CAR-T cells superior efficacy against solid tumors

Biochemical and immunological negative regulators converge to inhibit tumor-reactive Chimeric Antigen Receptor T (CAR-T) cells, which may explain clinical failures of CAR-T cell therapies against solid tumors. Here, we developed a multifaceted approach to genetically engineer allogeneic ( off -the-shelf) CAR-T cells resistant to both biochemical (adenosine) and immunological (PD-L1 and TGF-{beta}) inhibitory signaling. We multiplexed an adenine base editor with a CRISPR-Cas12b nuclease to manufacture a CAR-T cell product comprising six gene edits to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and resist major biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) immunosuppressive barriers in solid tumors. Combinatorial genetic disruption in CAR-T cells enabled superior anti-tumor efficacy leading to improved tumor elimination and survival in humanized mouse models that recapitulated the suppressive features of a human tumor microenvironment (TME). This novel engineering strategy conferred CAR-T cells resistance to a diverse TME, which may unlock the therapeutic potential of CAR-T cells against solid tumors. One Sentence SummaryMultiplex genome engineered CAR-T cells resistant to allorejection and the convergence of biochemical and immunological negative regulators within the tumor microenvironment exhibit superior efficacy against solid tumors.

cell biology↗

bioRxiv2022

An automated high-content synaptic phenotyping platform in human neurons and astrocytes reveals a role for BET proteins in synapse assembly

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth and cell viability, validating six small molecules which specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression programs. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

neuroscience↗

bioRxiv2019

Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional genomic studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design new fluorescently labeled piggyBac (PB) vectors to deliver robust and stable expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene and conduct a rigorous assessment of expression levels of the dCas9 effectors, gRNAs and targeted gene. Collectively, these data provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system would facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

molecular biology↗