SummaryGenerating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.

Key Points• Liver-targeted gene therapy for hemophilia can be improved by using computational promoter design in conjunction with hyperfunctional FIX.• Low and safe vector doses allow for stable supraphysiologic FIX that result in the induction of immune tolerance.The development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by using a computational strategy that increased factor IX (FIX) levels 11-to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyperfunctional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. IntroductionSignificant progress has recently been made toward the development of gene therapy for hemophilia B. Adenoassociated virus (AAV) vectors are among the most promising vectors for liver-directed gene therapy that are capable of achieving therapeutic factor IX (FIX) expression levels in patients suffering from severe hemophilia B. 1,2 Nevertheless, there are still some issues related to the induction of AAV capsidspecific T-cell-mediated immune response against the AAV-transduced cells that need to be addressed. [1][2][3][4] These inadvertent immune reactions curtailed long-term gene expression by eliminating the gene-modified cells and accounted for liver toxicity. Furthermore, the performance of these AAV vectors must be improved to achieve a bona fide cure. Consequently, there is a need to create the next-generation AAV vectors for liver-directed gene therapy that express higher FIX levels at lower vector doses, to the extent that stable physiologic levels of FIX can be attained, while preventing inadvertent AAV capsid-specific T-cell responses and liver toxicity. The availability of more potent vectors would also ease manufacturing needs. To increase the potency of AAV-FIX vectors, we explored the use of a bioinformatics algorithm that resulted in the identification of transcriptional cis-regulatory modules ( 5 These CRMs contained evolutionary conserved clusters of transcription factor binding-site motifs that confer high tissue-specific gene expression. We then combined these hepatocyte-specific CRMs (HS-CRMs) with a synthetic codon-optimized hyperfunctional FIX transgene (ie, Padua R338L) that conferred 15-fold higher expression and activity levels than its wild-type counterpart.6,7 This novel combination approach substantially reduced the dose requirement for reaching therapeutic efficacy and thus facilitates future scale-up and clinical translation. There is an Inside Blood Commentary on this article in this issue.The publication costs of this article were defrayed in part by page charge payment. T...

Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.

Gene therapy is a promising emerging therapeutic modality for the treatment of cardiovascular diseases and hereditary diseases that afflict the heart. Hence, there is a need to develop robust cardiac-specific expression modules that allow for stable expression of the gene of interest in cardiomyocytes. We therefore explored a new approach based on a genome-wide bioinformatics strategy that revealed novel cardiac-specific cis-acting regulatory modules (CS-CRMs). These transcriptional modules contained evolutionary-conserved clusters of putative transcription factor binding sites that correspond to a “molecular signature” associated with robust gene expression in the heart. We then validated these CS-CRMs in vivo using an adeno-associated viral vector serotype 9 that drives a reporter gene from a quintessential cardiac-specific α-myosin heavy chain promoter. Most de novo designed CS-CRMs resulted in a >10-fold increase in cardiac gene expression. The most robust CRMs enhanced cardiac-specific transcription 70- to 100-fold. Expression was sustained and restricted to cardiomyocytes. We then combined the most potent CS-CRM4 with a synthetic heart and muscle-specific promoter (SPc5-12) and obtained a significant 20-fold increase in cardiac gene expression compared to the cytomegalovirus promoter. This study underscores the potential of rational vector design to improve the robustness of cardiac gene therapy.

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in LMNA. The main proteins encoded by LMNA are Lamin A and C, which together with Lamin B1 and B2, form the nuclear lamina: a mesh-like structure located underneath the inner nuclear membrane. Laminopathies show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and adipose tissue, while others cause multisystem disease with accelerated aging. Although several pathogenic mechanisms have been proposed, the exact pathophysiology of laminopathies remains unclear, compounded by the rarity of these disorders and lack of easily accessible cell types to study. To overcome this limitation, we used induced pluripotent stem cells (iPSCs) from patients with skeletal muscle laminopathies such as LMNA-related congenital muscular dystrophy and limb-girdle muscular dystrophy 1B, to model disease phenotypes in vitro. iPSCs can be derived from readily accessible cell types, have unlimited proliferation potential and can be differentiated into cell types that would otherwise be difficult and invasive to obtain. iPSC lines from three skeletal muscle laminopathy patients were differentiated into inducible myogenic cells and myotubes. Disease-associated phenotypes were observed in these cells, including abnormal nuclear shape and mislocalization of nuclear lamina proteins. Nuclear abnormalities were less pronounced in monolayer cultures of terminally differentiated skeletal myotubes than in proliferating myogenic cells. Notably, skeletal myogenic differentiation of LMNA-mutant iPSCs in artificial muscle constructs improved detection of myonuclear abnormalities compared to conventional monolayer cultures across multiple pathogenic genotypes, providing a high-fidelity modeling platform for skeletal muscle laminopathies. Our results lay the foundation for future iPSC-based therapy development and screening platforms for skeletal muscle laminopathies.

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.

In vivo tissue-specific genome editing at the desired loci is still a challenge. Here, we report that AAV9-delivery of truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and sequence-specific targeting in the mouse factor IX (F9) gene. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing, and deep sequencing of on-target and putative off-target sites. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. Consequently, the insertions and deletion (indel) frequency was robust in the liver (up to 50%) in the desired target loci of the F9 gene, with no evidence of targeting in other organs or other putative off-target sites. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B. The in vivo efficacy of the truncated gRNA was as high as that of full-length gRNA. Cas9 expression was transient in neonates, representing an attractive "hit-and-run" paradigm. Our findings have potentially broad implications for somatic gene targeting in the liver using the CRISPR/Cas9 platform.

Skeletal muscle is a complex tissue composed of multinucleated myo bres responsible for force generation, supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function is instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are rst differentiated into myogenic, neural and vascular progenitor cells, and then combined within 3D hydrogels under tension to generate an aligned myo bre scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers.Importantly, bioengineered muscles provide a high-delity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 days, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro, as well as for in vivo studies, providing proof-ofprinciple of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.