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Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) has shown clear neurological benefit in rare diseases, which is achieved through the engraftment of genetically modified microglia-like cells (MLCs) in the brain. Still, the engraftment dynamics and the nature of engineered MLCs, as well as their potential use in common neurogenerative diseases, have remained largely unexplored. Here, we comprehensively characterized how different routes of administration affect the biodistribution of genetically engineered MLCs and other HSPC derivatives in mice. We generated a high-resolution single-cell transcriptional map of MLCs and discovered that they could clearly be distinguished from macrophages as well as from resident microglia by the expression of a specific gene signature that is reflective of their HSPC ontogeny and irrespective of their long-term engraftment history. Lastly, using murine models of Parkinson's disease and frontotemporal dementia, we demonstrated that MLCs can deliver therapeutically relevant levels of transgenic protein to the brain, thereby opening avenues for the clinical translation of HSPC-GT to the treatment of major neurological diseases.

Pompe disease is a rare genetic neuromuscular disorder caused by acid alpha-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy (ERT) is the current standard of care, which prolongs the quality of life for Pompe patients. However, ERT has limitations due to lack of enzyme penetration into the central nervous system (CNS) and skeletal muscles, immunogenicity against the recombinant enzyme, and requires life-long biweekly infusions. In a preclinical mouse model, a clinically relevant promoter to drive lentiviral vector-mediated expression of engineered GAA in autologous hematopoietic stem and progenitor cells (HSPC) was tested with nine unique human chimeric GAA coding sequences incorporating distinct peptide tags and codon-optimization iterations. Vectors including glycosylation independent lysosomal targeting (GILT) tags resulted in effective GAA enzyme delivery into key disease tissues with enhanced reduction of glycogen, myofiber and CNS vacuolation, compared to non-tagged GAA in Gaa knockout mice, a model of Pompe disease. Genetically modified microglial cells in brains were detected at low levels, but provided robust correction. Furthermore, an aminoacid substitution in the tag added to reduced capacity to induce insulin signaling and there was no evidence of off-target effects. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model providing a promising vector candidate for further investigation.One Sentence SummaryA candidate vector for hematopoietic stem cell gene therapy of Pompe disease.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) results in the engraftment of genetically modified microglia-like cells (MLCs) in the brain. While HSPC-GT has shown a clear neurological benefit in the clinic for specific rare diseases, the nature of MLC engraftment in the brain and the functional characteristics of MLCs remain contentious. Here we comprehensively characterized how different routes of administration affect the engraftment and biodistribution of genetically engineered HSPC-derivatives in mice. Using high-throughput single-cell profiling, we show that MLCs bear a transcriptional signature similar to resident microglia rather than invading macrophages. However, MLCs could clearly be distinguished from resident microglia by expression of a specific set of genes. Finally, in murine models of Parkinson’s disease and frontotemporal dementia, we demonstrate that MLCs can provide therapeutically relevant levels of protein to the brain, thereby potentially opening avenues of HSPC-GT to address the underlying disease etiology of these and other similar disorders.

We report the application of quantitative mass spectrometry to identify plasma membrane proteins differentially expressed in melanoma cells with high vs. low metastatic abilities. Using stable isotope labeling with amino acids in culture (SILAC) coupled with nanospray tandem mass spectrometry, we identified CUB-domain–containing protein 1 (CDCP1) as one such differentially expressed transmembrane protein. CDCP1 is not only a surface marker for cells with higher metastatic potential, but also functionally involved in enhancing tumor metastasis. Overexpression of CDCP1 also correlates with activation of Src. Pharmacological reagents, PP2 and Dasatinib, which block Src family kinase activation, blocked scattered growth of CDCP1-overexpressing cells in 3D Matrigel culture, suggesting that CDCP1 might function through the activation of Src-family kinases (SFKs). This hypothesis was further supported by mutational studies of CDCP1. Whereas wild-type CDCP1 enhances Src activation, point mutation Y734F abolishes in vitro dispersive growth in 3D culture and in vivo metastasis-enhancing activities of CDCP1. In addition, the Y734F mutation also eliminated enhanced Src activation. Thus, this work provides molecular mechanisms for the metastasis-enhancing functions of CDCP1.

Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells1, 2 and have been presumed to retain labels such as the nucleotide analogue 5-bromodeoxyuridine (BrdU). However, recently it has been demonstrated that BrdU-retention is neither sensitive nor specific for HSCs3. Here we show that transient, transgenic expression of a Histone2B (H2B)-Green Fluorescent Protein (GFP) fusion protein in mice allows superior labeling of HSCs and permits improved analysis of their turnover in combination with other markers. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a highly stringent marker combination (L−K+S+CD48−CD150+)4, revealed unexpected heterogeneity in their proliferation rates and suggests that ~ 20% of HSCs turn over at an extremely low rate (≤ 0.8–1.8% per day). Prospective isolation and transplantation of L−K+S+CD48−CD150+ HSCs with different H2B-GFP levels revealed that higher H2B-GFP label retention correlates with superior long-term repopulation potential.

Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion, as a single HSC may reconstitute lethally irradiated hosts1. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life2,3. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified4,5,6, transcription factors restricting their cycling have remained elusive. Here we report that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells7,8, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity.

The transcriptional coactivator Yes-associated protein (YAP) is a major regulator of organ size and proliferation in vertebrates. As such, YAP can act as an oncogene in several tissue types if its activity is increased aberrantly. Although no activating mutations in the yap1 gene have been identified in human cancer, yap1 is located on the 11q22 amplicon, which is amplified in several human tumors. In addition, mutations or epigenetic silencing of members of the Hippo pathway, which represses YAP function, have been identified in human cancers. Here we demonstrate that, in addition to increasing tumor growth, increased YAP activity is potently prometastatic in breast cancer and melanoma cells. Using a Luminex-based approach to multiplex in vivo assays, we determined that the domain of YAP that interacts with the TEAD/TEF family of transcription factors but not the WW domains or PDZ-binding motif, is essential for YAP-mediated tumor growth and metastasis. We further demonstrate that, through its TEAD-interaction domain, YAP enhances multiple processes known to be important for tumor progression and metastasis, including cellular proliferation, transformation, migration, and invasion. Finally, we found that the metastatic potential of breast cancer and melanoma cells is strongly correlated with increased TEAD transcriptional activity. Together, our results suggest that increased YAP/TEAD activity plays a causal role in cancer progression and metastasis.

Hematopoietic stem cells (HSCs) sustain blood formation throughout life. Pathways regulating maintenance of adult HSCs are largely unknown. Here we report that the Ets-related transcription factor Tel/Etv6, the product of a locus frequently involved in translocations in leukemia, is a selective regulator of HSC survival. Following inactivation of Tel/Etv6, HSCs are lost in the adult bone marrow but their progeny are unaffected and transiently sustain blood formation. Accordingly, absence of Tel/Etv6 after lineage commitment is ostensibly without consequence except for unexpected impairment of maturation of megakaryocytes. Thus, we establish Tel/Etv6 as a selective and essential regulator of postembryonic HSCs.

SummaryThe initial steps in the pathogenesis of acute leukemia remain incompletely understood. The TEL-AML1 gene fusion, the hallmark translocation in Childhood Acute Lymphoblastic Leukemia and the first hit, occurs years before the clinical disease, most often in utero. We have generated mice in which TEL-AML1 expression is driven from the endogenous promoter and can be targeted to specific populations. TEL-AML1 renders mice prone to malignancy after chemical mutagenesis when expressed in hematopoietic stem cells (HSCs), but not in early lymphoid progenitors. We reveal that TEL-AML1 markedly increases the number of HSCs and predominantly maintains them in the quiescent (G0) stage of the cell cycle. TEL-AML1+ HSCs retain self-renewal properties and contribute to hematopoiesis, but fail to out-compete normal HSCs. Our work shows that stem cells are susceptible to subversion by weak oncogenes that can subtly alter their molecular program to provide a latent reservoir for the accumulation of further mutations.