Explore the vast range of titles available.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy. Adeno-associated virus is the basis of many gene therapies and gene transfer vectors. Here the authors report a pipeline to enable side-by-side comparison of pre-selected capsids in a high throughput manner.

Zsuzsanna Izsvák and colleagues report the generation of a hyperactive version of the Sleeping Beauty transposase that supports efficient and stable gene transfer into human CD34 + cells enriched for hematopoietic stem or progenitor cells. The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with ∼100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35–50% stable gene transfer in human CD34 + cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34 + cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo . Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.

Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in mouse models has been achieved paving the way toward the first clinical trials. LVs can deliver genes ex vivo into bona fide stem cells, particularly hematopoietic stem cells, allowing for stable transgene expression upon hematopoietic reconstitution. They are also useful to generate induced pluripotent stem cells. LVs can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Targetable LVs can be generated by incorporating specific ligands or antibodies into the vector envelope. Immune responses toward the transgene products and transduced cells can be repressed using microRNA-regulated vectors. Though there are safety concerns regarding insertional mutagenesis, their integration profile seems more favorable than that of γ-retroviral vectors (γ-RVs). Moreover, it is possible to minimize this risk by modifying the vector design or by employing integration-deficient LVs. In conjunction with zinc-finger nuclease technology, LVs allow for site-specific gene correction or addition in predefined chromosomal loci. These recent advances underscore the improved safety and efficacy of LVs with important implications for clinical trials.

In this protocol, pluripotent stem cells are first differentiated into expandable cells resembling human mesoangioblasts; subsequently, transient MyoD induction drives differentiation into multinucleated myotubes. 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.

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.

The first gene therapy products for hemophilia A and B have recently been approved by the regulatory authorities. Although this is an important milestone for people with hemophilia, there is still a need to further improve on the efficacy, safety, and stability of expression and to ultimately include pediatric patients before the onset of arthropathy and other complications caused by uncontrolled bleeding. To overcome some of the limitations of conventional gene therapy strategies, gene editing is currently being explored in preclinical studies. Gene editing allows for targeted modifications of the human genome with unprecedented specificity based on zinc finger nuclease, meganuclease, transcription activator-like endonuclease, or clustered regularly interspaced short palindromic repeats (CRISPR) technologies that induce double-strand DNA breaks (DSB). Next-generation gene editing strategies, such as those dependent on CRISPR-derived base or prime editors, allow targeted genetic modification independent of the induction of DSBs, offering a potential safer alternative. Sustained efficacy and production of factor VIII or factor IX can be achieved after gene editing in patient-derived cells or in adult or newborn hemophilia A or B mouse models. These preclinical studies pave the way toward phase I/II clinical trials in patients with severe hemophilia. The potential risk of undesired off-target modifications of the human genome and adverse immune reactions, and the need for efficient delivery of the gene editing components, need to be rigorously addressed before the promise of gene editing for hemophilia can ultimately be fulfilled. CRISPR and gene editing technologies for bleeding disorders Gene therapy has recently made big steps forward, with the first approved treatments now available for people with hemophilia A and B. These treatments are promising, but there is still room to improve how well they work, how long they last, and how safe they are. Eventually, we want to be able to treat children early, before they start having joint damage and other bleeding-related problems. To make gene therapy even better, gene editing is being developed, a more precise way to fix faulty genes. This includes advanced tools like CRISPR, TALENs, ZFNs, and meganucleases, which can make small cuts in the DNA to help correct genetic mistakes. Newer methods, such as base editing and prime editing, can fix genes without cutting the DNA, which might be even safer. So far, gene editing has worked well in lab-grown cells from patients and in mouse models of hemophilia, showing it can restore the production of clotting factors (Factor VIII or IX). These early successes are helping to prepare for future human trials. However, there are still important challenges. We need to make sure the edits do not accidentally change the wrong parts of the DNA (called off-target effects), avoid immune system reactions, and find the best ways to deliver the gene editing tools to the right cells. Solving these problems is key before gene editing can become a routine treatment for hemophilia.

The human musculature is a promising and pivotal target for human gene therapy, owing to numerous diseases that affect this tissue and that are often monogenic, making them amenable to treatment and potentially cure on the genetic level. Particularly attractive would be the possibility to deliver clinically relevant DNA to muscle tissue from a minimally invasive, intravenous vector delivery. To date, this aim has been approximated by the use of Adeno-associated viruses (AAV) of different serotypes (rh.74, 8, 9) that are effective, but unfortunately not specific to the muscle and hence not ideal for use in patients. Here, we have thus studied the muscle tropism and activity of another AAV serotype, AAVpo1, that was previously isolated from pigs and found to efficiently transduce muscle following direct intramuscular injection in mice. The new data reported here substantiate the usefulness of AAVpo1 for muscle gene therapies by showing, for the first time, its ability to robustly transduce all major muscle tissues, including heart and diaphragm, from peripheral infusion. Importantly, in stark contrast to AAV9 that forms the basis for ongoing clinical gene therapy trials in the muscle, AAVpo1 is nearly completely detargeted from the liver, making it a very attractive and potentially safer option.

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA. Hemophilia A is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. Targeting FVIII expression in endothelial and myeloid cells, the natural site of its production, by lentiviral vector gene transfer, Follenzi et al. obtained therapeutic levels of FVIII activity without formation of neutralizing antibodies in hemophilic mice.

Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction including hard-to-transfect hematopoietic stem/progenitor cells (HSCs), and the most sensitive to OPI, underpinning the need for careful optimization of the transposon components. SB and PB were about equally active, and both more efficient than Tol2, under nonrestrictive conditions. All three systems provided long-term transgene expression in human cells with minimal signs of silencing. Indeed, mapping of Tol2 insertion sites revealed significant underrepresentation within chromosomal regions with H3K27me3 histone marks typically associated with transcriptionally repressed heterochromatin. SB, Tol2, and PB constitute complementary research tools for gene transfer in mammalian cells with important implications for fundamental and translational research.