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

Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to an accumulation of glycogen in lysosomes, and resulting in the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and has limitations such as poor penetration into skeletal muscle and both the central and peripheral nervous systems, a risk of immune responses against the recombinant enzyme, and the requirement 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 central nervous tissue samples to gain insights into the underlying mechanisms of phenotype correction.

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

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.

MEIS1 regulates an HLF-oxidative stress axis in MLL-fusion gene leukemia.

Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.

Meis1 preserves hematopoietic stem cells in mice by limiting oxidative stress.

The transcription factor Meis1 is expressed preferentially in hematopoietic stem cells (HSCs) and overexpressed in certain leukemias. However, the functions of Meis1 in hematopoiesis remain largely unknown. In the present study, we found that Meis1 is required for the maintenance of hematopoiesis under stress and over the long term, whereas steady-state hematopoiesis was sustained in the absence of Meis1 in inducible knock-out mice. BM cells of Meis1-deficient mice showed reduced colony formation and contained significantly fewer numbers of long-term HSCs, which exhibited loss of quiescence. Further, we found that Meis1 deletion led to the accumulation of reactive oxygen species in HSCs and decreased expression of genes implicated in hypoxia response. Finally, reactive oxygen species scavenging by N-acetyl cysteine or stabilization of hypoxia signaling by knockdown of the von-Hippel-Lindau (VHL) protein led to reversal of the effects of Meis1 deletion. The results of the present study demonstrate that Meis1 protects and preserves HSCs by restricting oxidative metabolism.

Gene Therapy Developments for Pompe Disease.

Pompe disease is an inherited neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The most severe form is infantile-onset Pompe disease, presenting shortly after birth with symptoms of cardiomyopathy, respiratory failure and skeletal muscle weakness. Late-onset Pompe disease is characterized by a slower disease progression, primarily affecting skeletal muscles. Despite recent advancements in enzyme replacement therapy management several limitations remain using this therapeutic approach, including risks of immunogenicity complications, inability to penetrate CNS tissue, and the need for life-long therapy. The next wave of promising single therapy interventions involves gene therapies, which are entering into a clinical translational stage. Both adeno-associated virus (AAV) vectors and lentiviral vector (LV)-mediated hematopoietic stem and progenitor (HSPC) gene therapy have the potential to provide effective therapy for this multisystemic disorder. Optimization of viral vector designs, providing tissue-specific expression and GAA protein modifications to enhance secretion and uptake has resulted in improved preclinical efficacy and safety data. In this review, we highlight gene therapy developments, in particular, AAV and LV HSPC-mediated gene therapy technologies, to potentially address all components of the neuromuscular associated Pompe disease pathology.

Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease.

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. 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 promising vector candidates for further investigation.

Aryl Hydrocarbon Receptor Deficiency in an Exon 3 Deletion Mouse Model Promotes Hematopoietic Stem Cell Proliferation and Impacts Endosteal Niche Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor belonging to the Per-Arnt-Sim (PAS) family of proteins. The AHR is involved in hematopoietic stem cell (HSC) functions including self-renewal, proliferation, quiescence, and differentiation. We hypothesize that AHR impacts HSC functions by influencing genes that have roles in HSC maintenance and function and that this may occur through regulation of bone marrow (BM) niche cells. We examined BM and niche cells harvested from 8-week-old AHR null-allele (KO) mice in which exon 3 was deleted in the Ahr gene and compared these data to cells from B6 control mice; young and old (10 months) animals were also compared. We report changes in HSCs and peripheral blood cells in mice lacking AHR. Serial transplantation assays revealed a significant increase in long term HSCs. There was a significant increase in mesenchymal stem cells constituting the endosteal BM niche. Gene expression analyses of HSCs revealed an increase in expression of genes involved in proliferation and maintenance of quiescence. Our studies infer that loss of AHR results in increased proliferation and self-renewal of long term HSCs, in part, by influencing the microenvironment in the niche regulating the balance between quiescence and proliferation in HSCs.

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells.

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice.