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Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration. Progranulin deficiency causes untreatable neurodegenerative diseases. Here, the authors show that hematopoietic stem cell transplantation and optimized brain conditioning correct the disease phenotype in progranulin-deficient mice.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool. A small molecule enhances targeted gene integration at therapeutically relevant loci in human primary cells.

Developing new fluorophores for advanced imaging techniques requires exploring new chemical space. While generative AI approaches have shown promise in designing novel dye scaffolds, prior efforts often produced synthetically intractable candidates due to a lack of reaction constraints. Here, we developed SyntheFluor-RL, a generative AI model that employs known reaction libraries and molecular building blocks to create readily synthesizable fluorescent molecule scaffolds via reinforcement learning. To guide the generation of fluorophores, SyntheFluor-RL employs a scoring function built on multiple graph neural networks (GNNs) that predict key photophysical properties, including photoluminescence quantum yield, absorption, and emission wavelengths. These outputs are dynamically weighted and combined with a computed pi-conjugation score to prioritize candidates with desirable optical characteristics and synthetic feasibility. SyntheFluor-RL generated 11,590 candidate molecules, which were filtered to 19 structures predicted to possess dye-like properties. Of the 19 molecules, 14 were synthesized and 13 were experimentally confirmed. The top three were characterized, with the lead compound featuring a benzothiadiazole chromophore and exhibiting strong fluorescence (PLQY = 0.62), a large Stokes shift (97 nm), and a long excited-state lifetime (11.5 ns). These results demonstrate the effectiveness of SyntheFluor-RL in the identification of synthetically accessible fluorophores for further development.