Biology subjects

Malkov, D.

Publications and source records attributed to Malkov, D..

3 recordsLinked to original sources

bioRxiv2026

End-To-End, Fully Automated Manufacturing of Hematopoietic Stem and Progenitor Cell Therapy Via CCR5-Targeted Integration

Gene therapies utilizing hematopoietic stem and progenitor cells (HSPCs) have curative potential across hematologic diseases, but manual manufacturing workflows are costly and difficult to scale, limiting patient access. Here, we demonstrated automation of an HSPC gene knock-in protocol on a high-throughput manufacturing platform designed for clinical-scale production. The platform executed all steps, from post-thaw HSPC inoculation through Cas9 electroporation, AAV6 transduction, and harvest, producing gene-edited HSPCs (geHSPCs) carrying an anti-HIV antibody transgene at the CCR5 locus with high purity, preserved differentiation potential, and editing efficiencies comparable to small-scale processing. Following transplantation into mice, geHSPCs engrafted and reconstituted multiple hematopoietic lineages, demonstrating that repopulation capacity was preserved. This work shows that clinical-scale doses of geHSPCs meeting release criteria can be produced on an automated, high-throughput platform, while preserving key product attributes and in vivo function, providing a path toward scalable and cost-effective manufacturing that could broaden access to HSPC gene therapies.

genetics↗

bioRxiv2020

Tales of 1,008 Small Molecules: Phenomic Profiling through Live-cell Imaging in a Panel of Reporter Cell Lines

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC [≥]0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current studys conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.

pharmacology and toxicology↗

bioRxiv2017

Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression

Targeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integration of donor ssDNA. Most importantly, the ratio of ssDNA donor integration to non-homologous end joining (NHEJ) was observed to increase, suggesting that cell cycle control Cas9 expression may be an effective strategy to bias DNA repair outcomes.

molecular biology↗