High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing

Plasschaert, Robert N.; DeAndrade, Mark P.; Hull, Fritz; Nguyen, Quoc; Peterson, Tara; Yan, Aimin; Loperfido, Mariana; Baricordi, Cristina; Barbarossa, Luigi; Yoon, John K.; Dogan, Yildirim; Unnisa, Zeenath; Schindler, Jeffrey W.; Til, Niek P. van; Mason, Chris

doi:10.1016/j.ymthe.2022.05.022

Cited by 12 publications

(9 citation statements)

References 61 publications

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“…One of the biggest challenges in treating inborn metabolic diseases using enzyme cross- complementation is the delivery of the missing enzyme to the brain. HSPC gene therapy could be indicated for this application, because cells derived from HSPCs can cross the blood brain barrier and establish in the brain 19 . We thus evaluated whether this was true for CD11b edited HSPCs in our in vivo cohorts.…”

Section: Resultsmentioning

confidence: 99%

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Seclen,

Jang,

Lawal

et al. 2024

Preprint

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Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoter-less intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron ofCD11band observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages.In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUAex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

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Section: Resultsmentioning

confidence: 99%

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Seclen,

Jang,

Lawal

et al. 2024

Preprint

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“…(25, 34, 35) An advantage of the followed approach using HSPC transplantation is that biodistribution of genetically modified cells is broad throughout the body and includes effective delivery to the CNS. (36) Furthermore, the MND promoter showed robust expression in microglia-like cells in the brain compared to two commonly used house-keeping gene promoters for HSC gene therapy, the EFS and PGK promoter. However, we only compared expression differences between the promoter variants with GFP as a model transgene.…”

Section: Discussionmentioning

confidence: 99%

“…All qPCR assays consisted of oligonucleotide primers and probe mixes containing either a TaqMan 6-carboxyfluorescenin (FAM) or VIC TM fluorescent probe designed to amplify the HIV Psi vector sequence or housekeeping genes glycosyltransferase like domain containing 1 ( Gtdc1 ) or transferrin receptor protein 1 ( Tfrc ) as previously described. (14, 36) For qPCR VCN assessment, a single plasmid containing both sequences was used as a reference standard in a range of 50 to 5 ξ 10 7 copies. Data was reported as VCN/diploid genome.…”

Section: Methodsmentioning

confidence: 99%

Preclinical lentiviral vector-mediated hematopoietic stem and progenitor cell gene therapy corrects Pompe disease-related muscle and neurological manifestations

Yoon,

Schindler,

Loperfido

et al. 2023

Preprint

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Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to the accumulation of glycogen in lysosomes and the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and demonstrates poor penetration into skeletal muscle and the central and peripheral nervous systems, susceptibility to immune responses against the recombinant enzyme, and the need for high doses and frequent infusions. To overcome these limitations, lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy has been proposed as a next-generation approach for treating Pompe disease. This study demonstrates the potential of lentiviral HSPC gene therapy to reverse the pathological effects of Pompe disease in a preclinical mouse model. It includes a comprehensive safety assessment via integration site analysis, along with single-cell RNA sequencing analysis of CNS samples to gain insights into the underlying mechanisms of phenotype correction.One Sentence Summary:Preclinical hematopoietic stem cell gene therapy for the treatment of Pompe disease.

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“…While transcriptional signatures that separate MGLCs and microglia have been described 18,70,71 , the single-cell heterogeneity of MGLCs engrafted in the brain following conditioning with BU/CSF1Ri has not been characterized. To e x a m i n e t h i s , w e performed single-cell RNA sequencing (scRNA-seq) of FACS-sorted CD45+ CD11b+ cells isolated from mice that underwent BU + PLX and BMT and naïve mice.…”

Section: Mglcs Are Heterogeneous and Express Microglia-specific Genesmentioning

confidence: 99%

CNS Repopulation by Hematopoietic-Derived Microglia-Like Cells Corrects Progranulin deficiency

Gomez-Ospina,

Colella,

Sayana

et al. 2023

Preprint

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Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn-/- mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.

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High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing

Cited by 12 publications

References 61 publications

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Preclinical lentiviral vector-mediated hematopoietic stem and progenitor cell gene therapy corrects Pompe disease-related muscle and neurological manifestations

CNS Repopulation by Hematopoietic-Derived Microglia-Like Cells Corrects Progranulin deficiency

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