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Abstract Heart failure is a common and deadly disease requiring new treatments. The neuregulin-1/ERBB4 pathway offers cardioprotective benefits, but using recombinant neuregulin-1 as therapy has limitations due to the need for intravenous delivery and lack of receptor specificity. We hypothesize that small-molecule activation of ERBB4 could protect against heart damage and fibrosis. To test this, we conduct a screening of 10,240 compounds and identify eight structurally similar ones (EF-1 to EF-8) that induce ERBB4 dimerization, with EF-1 being the most effective. EF-1 reduces cell death and hypertrophy in cardiomyocytes and decreases collagen production in cardiac fibroblasts in an ERBB4-dependent manner. In wild-type mice, EF-1 inhibits angiotensin-II-induced fibrosis in males and females and reduces heart damage caused by doxorubicin and myocardial infarction in females, but not in Erbb4-null mice. This study shows that small-molecule ERBB4 activation is feasible and may lead to a novel class of drugs for treating heart failure.

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective Kv11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations.

Aim: Channelrhodopsins (ChRs) are a large family of light-gated ion channels with distinct properties, which is of great importance in the selection of a ChR variant for a given application. However, data to guide such selection for cardiac optogenetic applications are lacking. Therefore, we investigated the functioning of different ChR variants in normal and pathological hypertrophic cardiomyocytes subjected to various illumination protocols.Methods and Results: Isolated neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with lentiviral vectors to express one of the following ChR variants: H134R, CatCh, ReaChR, or GtACR1. NRVMs were treated with phenylephrine (PE) to induce pathological hypertrophy (PE group) or left untreated [control (CTL) group]. In these groups, ChR currents displayed unique and significantly different properties for each ChR variant on activation by a single 1-s light pulse (1 mW/mm2: 470, 565, or 617 nm). The concomitant membrane potential (Vm) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average Vm during illumination (Vplateau: −38 mV) as compared with a Vplateau > −20 mV for the other ChR variants. On repetitive activation at increasing frequencies (10-ms pulses at 1–10 Hz for 30 s), peak currents, which are important for cardiac pacing, decreased with increasing activation frequencies by 17–78% (p < 0.05), while plateau currents, which are critical for arrhythmia termination, decreased by 10–75% (p < 0.05), both in a variant-specific manner. In contrast, the corresponding Vplateau remained largely stable. Importantly, current properties and Vm responses were not statistically different between the PE and CTL groups, irrespective of the variant used (p > 0.05).Conclusion: Our data show that ChR variants function equally well in cell culture models of healthy and pathologically hypertrophic myocardium but show strong, variant-specific use-dependence. This use-dependent nature of ChR function should be taken into account during the design of cardiac optogenetic studies and the interpretation of the experimental findings thereof.

The majority of disease-associated genetic variants lie in non-coding regions. Here the authors generated and compiled human transcriptomic, epigenomic and chromatin conformation datasets, to identify genes associated with atrial fibrillation and functional non-coding variants.

Abstract Background Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown. Methods To investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein. Results Tbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process. Conclusion The present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells.

Abstract Background Mesenchymal stem cells (MSCs) are attractive choices in regenerative medicine and can be genetically modified to obtain better results in therapeutics. Bone development and metabolism are controlled by various factors including microRNAs (miRs) interference, which are small non-coding endogenous RNAs. Methods In the current study, the effects of forced miR-148b expression was evaluated on osteogenic activity. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with bicistronic lentiviral vector encoding hsa-miR-148b-3p or -5p and the enhanced green fluorescent protein. Fourteen days post-transduction, immunostaining as well as Western blotting were used to analyze osteogenesis. Results Overexpression of miR-148b-3p increased the osteogenic differentiation of human BM-MSCs as demonstrated by anenhancement of mineralized nodular formation and an increase in the levels of osteoblastic differentiation biomarkers, alkaline phosphatase and collagen type I. Conclusions Since lentivirally overexpressed miR-148b-3p increased osteogenic differentiation capability of BM-MSCs, this miR could be applied as a therapeutic modulator to optimize bone function.

Atrial fibrillation (AF) is accompanied by a detrimental loss of functional cardiomyocytes. Here, Zhang et al. show that AF-induced cardiomyocyte dysfunction is a consequence of DNA damage-mediated PARP1 activation, which leads to depletion of NAD+ and further oxidative stress and DNA damage, and identify PARP1 inhibition as a potential therapeutic strategy in the treatment of AF.

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.

Rationale: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. Aim: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. Methods and results: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. Conclusion: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.

The generation of abnormal excitations in pathological regions of the heart is a main trigger for lethal cardiac arrhythmias. Such abnormal excitations, also called ectopic activity, often arise from areas with local tissue heterogeneity or damage accompanied by localized depolarization. Finding the conditions that lead to ectopy is important to understand the basic biophysical principles underlying arrhythmia initiation and might further refine clinical procedures. In this study, we are the first to address the question of how geometry of the abnormal region affects the onset of ectopy using a combination of experimental, in silico, and theoretical approaches. We paradoxically find that, for any studied geometry of the depolarized region in optogenetically modified monolayers of cardiac cells, primary ectopic excitation originates at areas of maximal curvature of the boundary, where the stimulating electrotonic currents are minimal. It contradicts the standard critical nucleation theory applied to nonlinear waves in reaction-diffusion systems, where a higher stimulus is expected to produce excitation more easily. Our in silico studies reveal that the nonconventional ectopic activity is caused by an oscillatory instability at the boundary of the damaged region, the occurrence of which depends on the curvature of that boundary. The onset of this instability is confirmed using the Schrödinger equation methodology proposed by Rinzel and Keener [SIAM J. Appl. Math. 43, 907 (1983)SMJMAP0036-139910.1137/0143058]. Overall, we show distinctively novel insight into how the geometry of a heterogeneous cardiac region determines ectopic activity, which can be used in the future to predict the conditions that can trigger cardiac arrhythmias.

Cardiovascular disease is the leading cause of death globally, and stem cell therapy remains one of the most promising strategies for regeneration or repair of the damaged heart. We report that human placenta-derived multipotent cells (hPDMCs) can modulate cardiac injury in small and large animal models of myocardial ischemia (MI) and elucidate the mechanisms involved. We found that hPDMCs can undergo in vitro cardiomyogenic differentiation when cocultured with mouse neonatal cardiomyocytes. Moreover, hPDMCs exert strong proangiogenic responses in vitro toward human endothelial cells mediated by secretion of hepatocyte growth factor, growth-regulated oncogene-α, and interleukin-8. To test the in vivo relevance of these results, small and large animal models of acute MI were induced in mice and minipigs, respectively, by permanent left anterior descending (LAD) artery ligation, followed by hPDMC or culture medium-only implantation with follow-up for up to 8 weeks. Transplantation of hPDMCs into mouse heart post-acute MI induction improved left ventricular function, with significantly enhanced vascularity in the cell-treated group. Furthermore, in minipigs post-acute MI induction, hPDMC transplantation significantly improved myocardial contractility compared to the control group ( p =0.016) at 8 weeks postinjury. In addition, tissue analysis confirmed that hPDMC transplantation induced increased vascularity, cardiomyogenic differentiation, and antiapoptotic effects. Our findings offer evidence that hPDMCs can modulate cardiac injury in both small and large animal models, possibly through proangiogenesis, cardiomyogenesis, and suppression of cardiomyocyte apoptosis. Our study offers mechanistic insights and preclinical evidence on using hPDMCs as a therapeutic strategy to treat severe cardiovascular diseases.

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage, and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue, and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM), and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity, and differentiation potential. The division rate of AT-derived MSCs (AT-MSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM-, or SM-MSCs previously transduced with a lentiviral vector encoding β-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific β-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-based treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential, and because their harvesting is less demanding than that of BM- or SM-MSCs.

Mesenchymal stromal cells (MSCs) are attractive for cellular therapy of muscular dystrophies as they are easy to procure, can be greatly expanded ex vivo, and contribute to skeletal muscle repair in vivo. However, detailed information about the contribution of bone marrow (BM)-derived human MSCs (BM-hMSCs) to skeletal muscle regeneration in vivo is very limited. Here, we present the results of a comprehensive study of the fate of LacZ -tagged BM-hMSCs following implantation in cardiotoxin (CTX)-injured tibialis anterior muscles (TAMs) of immunodeficient mice. β-Galactosidase-positive (β-gal + ) human-mouse hybrid myofibers (HMs) were counted in serial cross sections over the full length of the treated TAMs of groups of mice at monthly intervals. The number of human cells was estimated using chemiluminescence assays. While the number of human cells declined gradually to about 10% of the injected cells at 60 days after transplantation, the number of HMs increased from day 10 onwards, reaching 104 ± 39.1 per TAM at 4 months postinjection. β-gal + cells and HMs were distributed over the entire muscle, indicating migration of the former from the central injection site to the ends of the TAMs. The identification of HMs that stained positive for human spectrin suggests myogenic reprogramming of hMSC nuclei. In summary, our findings reveal that BM-hMSCs continue to participate in the regeneration/remodeling of CTX-injured TAMs, resulting in ±5% HMs at 4 months after damage induction. Moreover, donor-derived cells were shown to express genetic information, both endogenous and transgenic, in recipient myofibers.

Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS-) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS- both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS- while at low acceptor-to-donor cell ratios FLPeNLS- was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed.

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

BackgroundMesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected.Methodology/principal findingsWe aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation.Conclusions/significanceOur findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable.

Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and conditional gene manipulation studies in vivo.

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).