Success Stories and Challenges Ahead in Hematopoietic Stem Cell Gene Therapy: Hemoglobinopathies as Disease Models.

Gene therapy is a relatively novel field that amounts to around four decades of continuous growth with its good and bad moments. Currently, the field has entered the clinical arena with the ambition to fulfil its promises for a permanent fix of incurable genetic disorders. Hemoglobinopathies as target diseases and hematopoietic stem cells (HSCs) as target cells of genetic interventions had a major share in the research effort toward efficiently implementing gene therapy. Dissection of HSC biology and improvements in gene transfer and gene expression technologies evolved in an almost synchronous manner to a point where the two fields seem to be functionally intercalated. In this review, we focus specifically on the development of gene therapy for hemoglobin disorders and look at both gene addition and gene correction strategies that may dominate the field of HSC-directed gene therapy in the near future and transform the therapeutic landscape for genetic diseases.

The role of small molecules in cell and gene therapy.

Cell and gene therapies have achieved impressive results in the treatment of rare genetic diseases using gene corrected stem cells and haematological cancers using chimeric antigen receptor T cells. However, these two fields face significant challenges such as demonstrating long-term efficacy and safety, and achieving cost-effective, scalable manufacturing processes. The use of small molecules is a key approach to overcome these barriers and can benefit cell and gene therapies at multiple stages of their lifecycle. For example, small molecules can be used to optimise viral vector production during manufacturing or used in the clinic to enhance the resistance of T cell therapies to the immunosuppressive tumour microenvironment. Here, we review current uses of small molecules in cell and gene therapy and highlight opportunities for medicinal chemists to further consolidate the success of cell and gene therapies.

The Landscape of Early Clinical Gene Therapies outside of Oncology.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

Vaccination of Macaques with DNA Followed by Adenoviral Vectors Encoding Simian Immunodeficiency Virus (SIV) Gag Alone Delays Infection by Repeated Mucosal Challenge with SIV.

Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomolgus macaques (MCM), Macaca fascicularis, vaccinated with unmodified SIV gag alone in a DNA prime followed by an rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low-dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection, a significant reduction in peak virus load was observed in all vaccinated animals, including those vaccinated with modified gag These findings suggest that a nonpersistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV type 1 (HIV-1) infection. The mechanisms are probably distinct from those of antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus-infected cells and may be mediated in part by CD4 T cells.IMPORTANCE The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.

Nonintegrating Gene Therapy Vectors.

Gene delivery vectors that do not rely on host cell genome integration offer several advantages for gene transfer, chiefly the avoidance of insertional mutagenesis and position effect variegation. However, unless engineered for replication and segregation, nonintegrating vectors will dilute progressively in proliferating cells, and are not exempt of epigenetic effects. This article provides an overview of the main nonintegrating viral (adenoviral, adeno-associated viral, integration-deficient retro-lentiviral, poxviral), and nonviral (plasmid vectors, artificial chromosomes) vectors used for preclinical and clinical cell and gene therapy applications. Particular emphasis is placed on their use in hematologic disease.

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

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.

Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies.

Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

Genome-wide computational analysis reveals cardiomyocyte-specific transcriptional Cis-regulatory motifs that enable efficient cardiac gene therapy.

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.

Fusion of ubiquitin to HIV gag impairs human monocyte-derived dendritic cell maturation and reduces ability to induce gag T cell responses.

The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs.

Triple trans-splicing adeno-associated virus vectors capable of transferring the coding sequence for full-length dystrophin protein into dystrophic mice.

Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1 kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7 kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying "in tandem" sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

Fragmentation of SIV-gag vaccine induces broader T cell responses.

BACKGROUND: High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition. METHODOLOGY/PRINCIPAL FINDINGS: three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector. CONCLUSION/SIGNIFICANCE: Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses.

Highly potent delivery method of gp160 envelope vaccine combining lentivirus-like particles and DNA electrotransfer.

Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like particles (VLPs) and the simplicity of use of DNA vaccines. Characterisation of lentivirus-like particles (lentiVLPs) by western blot, dynamic light scattering and electron microscopy revealed that their protein pattern, size and structure make them promising candidates for HIV-1 vaccines. Although all particles were similar with regard to size and distribution, they clearly differed in p24 capsid protein content suggesting that Rev may be required for particle maturation and Gag processing. In vivo, lentiVLP pseudotyping with the gp160 envelope or with a combination of gp160 and VSV-G envelopes did not influence the magnitude of the immune response but the combination of lentiVLPs with Alum adjuvant resulted in a more potent response. Interestingly, the strongest immune response was obtained when plasmids encoding lentiVLPs were co-delivered to mice muscles by electrotransfer, suggesting that lentiVLPs were efficiently produced in vivo or the packaging genes mediate an adjuvant effect. DNA electrotransfer of plasmids encoding lentivirus-like particles offers many advantages and appears therefore as a promising delivery method of HIV-1 vaccines.

Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8(+) T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. METHODS: We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. RESULTS: Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8 weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. CONCLUSIONS: The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD.

Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice.

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

ß-catenin as a potential key target for tumor suppression.

ß-catenin is a multifunctional protein identified to be pivotal in embryonic patterning, organogenesis and adult homeostasis. It plays a critical structural role in mediating cadherin junctions and is also an essential transcriptional co-activator in the canonical Wnt pathway. Evidence has been documented that both the canonical Wnt pathway and cadherin junctions are deregulated or impaired in a plethora of human malignancies. In the light of this, there has been a recent surge in elucidating the mechanisms underlying the etiology of cancer development from the perspective of ß-catenin. Here, we focus on the emerging roles of ß-catenin in the process of tumorigenesis by discussing novel functions of old players and new proteins, mechanisms identified to mediate or interact with ß-catenin and the most recently unraveled clinical implications of ß-catenin regulatory pathways toward tumor suppression.

Transcription factor rational design improves directed differentiation of human mesenchymal stem cells into skeletal myocytes.

There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.