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There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy. Adeno-associated viral vectors (AAV) are being developed for gene therapy of skeletal muscle, but it is a challenge to achieve robust gene expression. Here, the authors identify muscle-specific cisregulatory elements that lead to a substantial increase in micro-dystrophin and follistatin expression, resulting in a safe and sustainable rescue of the dystrophic phenotype in mouse models. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of interests All authors were former employees of AVROBIO, Inc., Cambridge, MA, USA during the conception and writing of the manuscript, except V.M., M.R., and A.S. N.P.v.T. and C.M. are inventors on patents in the field of HSC gene therapy. AVROBIO, Inc., has a preclinical gene therapy program for Pompe disease (AVR-RD-03) based on a genetically modified HSPC platform using lentiviral vectors. Collection of data and analysis was performed as part of the program. This research received no external funding and was sponsored by AVROBIO, Inc., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Integration site (IS) analysis is a fundamental analytical platform for evaluating the safety and efficacy of viral vector based preclinical and clinical Gene Therapy (GT). A handful of groups have developed standardized bioinformatics pipelines to process IS sequencing data, to generate reports, and/or to perform comparative studies across different GT trials. Keeping up with the technological advances in the field of IS analysis, different computational pipelines have been published over the past decade. These pipelines focus on identifying IS from single-read sequencing or paired-end sequencing data either using read-based or using sonication fragment-based methods, but there is a lack of a bioinformatics tool that automatically includes unique molecular identifiers (UMI) for IS abundance estimations and allows comparing multiple quantification methods in one integrated pipeline., Results: Here we present IS-Seq a bioinformatics pipeline that can process data from paired-end sequencing of both old restriction sites-based IS collection methods and new sonication-based IS retrieval systems while allowing the selection of different abundance estimation methods, including read-based, Fragment-based and UMI-based systems., Conclusions: We validated the performance of IS-Seq by testing it against the most popular  analytical workflow available in the literature (INSPIIRED) and using different scenarios. Lastly, by performing extensive simulation studies and a comprehensive wet-lab assessment of our IS-Seq pipeline we could show that in clinically relevant scenarios, UMI quantification provides better accuracy than the currently most widely used sonication fragment counts as a method for IS abundance estimation., (© 2023. The Author(s).)

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., Competing Interests: Declaration of interests All authors are current employees of AVROBIO, Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Hematopoietic Stem/Progenitor cells (HSPCs) are endowed with the role of maintaining a diverse pool of blood cells throughout the human life. Despite recent efforts, the nature of the early cell fate decisions remains contentious. Using single-cell RNA-Seq, we show that existing approaches to stratify bone marrow CD34+ cells reveal a hierarchically-structured transcriptional landscape of hematopoietic differentiation. Still, this landscape misses important early fate decisions. We here provide a broader transcriptional profiling of bone marrow lineage negative hematopoietic progenitors that recovers a key missing branchpoint into basophils and expands our understanding of the underlying structure of early adult human haematopoiesis. We also show that this map has strong similarities in topology and gene expression to that found in mouse. Finally, we identify the sialomucin CD164, as a reliable marker for the earliest branches of HSPCs specification and we showed how its use can foster the design of alternative transplantation cell products.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Skeletal muscle is the most abundant human tissue; therefore, an unlimited availability of myogenic cells has applications in regenerative medicine and drug development. Here we detail a protocol to derive myogenic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, and we also provide evidence for its extension to human iPS cells cultured without feeder cells. The procedure, which does not require the generation of embryoid bodies or prospective cell isolation, entails four stages with different culture densities, media and surface coating. Pluripotent stem cells are disaggregated to single cells and then differentiated into expandable cells resembling human mesoangioblasts. Subsequently, transient Myod1 induction efficiently drives myogenic differentiation into multinucleated myotubes. Cells derived from patients with muscular dystrophy and differentiated using this protocol have been genetically corrected, and they were proven to have therapeutic potential in dystrophic mice. Thus, this platform has been demonstrated to be amenable to gene and cell therapy, and it could be extended to muscle tissue engineering and disease modeling.

Human pluripotent stem cells represent a unique source for cell-based therapies and regenerative medicine. The intrinsic features of these cells such as their easy accessibility and their capacity to be expanded indefinitely overcome some limitations of conventional adult stem cells. Furthermore, the possibility to derive patient-specific induced pluripotent stem (iPS) cells in combination with the current development of gene modification methods could be used for autologous cell therapies of some genetic diseases. In particular, muscular dystrophies are considered to be a good candidate due to the lack of efficacious therapeutic treatments for patients to date, and in view of the encouraging results arising from recent preclinical studies. Some hurdles, including possible genetic instability and their efficient differentiation into muscle progenitors through vector/transgene-free methods have still to be overcome or need further optimization. Additionally, engraftment and functional contribution to muscle regeneration in pre-clinical models need to be carefully assessed before clinical translation. This review offers a summary of the advanced methods recently developed to derive muscle progenitors from pluripotent stem cells, as well as gene therapy by gene addition and gene editing methods using ZFNs, TALENs or CRISPR/Cas9. We have also discussed the main issues that need to be addressed for successful clinical translation of genetically corrected patient-specific pluripotent stem cells in autologous transplantation trials for skeletal muscle disorders.

Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

beta 2-Microglobulin (beta 2m) is a small polypeptide of 99 amino acids with a molecular weight of 11.800. It is found in serum, on the surface of almost all mammalian cells and is part of the surface antigen of the cell membrane of human lymphocytes. Moreover, the heavy chain of major histocompatibility complex class I molecules normally associates in the membrane with beta 2m. According to Canalis, et al this polypeptide is a bone derived growth factor. In our study, serum beta 2m, osteocalcin bone Gla-protein (BGP) and calcium phosphorous metabolism were evaluated in a group of 18 osteoporotic women. Nine were treated with an anabolic steroid, DecaDurabolin and 9 with salmon calcitonin (sCT) for 3 months. The same variables were evaluated in a control group (6 osteoporotic women) treated with oral Ca (1,500 mg/day for 3 months). A significant increase in serum beta 2m and BGP (p < 0.001) was observed after anabolic steroid while a significant decrease in serum beta 2m and BGP (p < 0.01) was observed after sCT. No significant variation for serum beta 2m and BGP was observed in the control group. No significant variation of calcium-phosphorous metabolism was observed in either the control group or the treated group. BGP variations suggest that anabolic steroid stimulates bone formation while a decrease in BGP observed after sCT suggests that this hormone depresses bone turnover via a block in osteoclast activity. Moreover it could be suggested that anabolic steroid and sCT affect beta 2m producing cells.

Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.