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Background: Duchenne muscular dystrophy (DMD) is a severe monogenic hereditary disease with early manifestation and a progressive course. Treatment options have so far been limited. Gene therapy opens up new options for DMD patients., Objectives: Against the background of a further death following DMD gene therapy, the side effects and risks of the gene therapeutics already approved or undergoing clinical trials will be evaluated and alternative gene therapeutics will be described. Based thereon, the future of DMD gene therapy will be discussed., Current Data: For the first time, in June 2023, delandistrogene moxeparvovec (SRP-9001), a gene replacement therapy based on an adeno-associated virus (AAV) vector, was approved in the USA for children aged 4-5 years with DMD. Other promising gene therapies are in preclinical development or clinical trials, including CRISPR/Cas9-mediated strategies to restore dystrophin expression. Two deaths following DMD gene therapy with high-dose AAV vectors were attributed to AAV-mediated immune responses. The pre-existing disease underlying the therapy is most likely involved in the fatal AAV toxicity., Conclusions: Although gene therapy applications of AAV vectors are generally considered safe, the systemic administration of high vector doses can lead to severe side effects with a potentially fatal outcome in individual patients, especially after activation of the immune system. In the future, new methods for immunosuppression, reduction of AAV dose and alternative vectors will therefore increasingly come to the fore., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia., (© 2021 by The American Society of Hematology.)

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C 4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C 4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 10 6 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.

HIV-1 infection is a complex, multi-step process involving not only viral, but also multiple cellular factors. To date, drug discovery methods have primarily focused on the inhibition of single viral proteins. We present an efficient and unbiased approach, compatible with biosafety level 1 (BSL-1) conditions, to identify inhibitors of HIV-1 reverse transcription, intracellular trafficking, nuclear entry and genome integration. Starting with a fluorescent assay setup, we systematically improved the screening methodology in terms of stability, efficiency and pharmacological relevance. Stability and throughput were optimized by switching to a luciferase-based readout. BSL-1 compliance was achieved without sacrificing pharmacological relevance by using lentiviral particles pseudo-typed with the mouse ecotropic envelope protein to transduce human PM1 T cells gene-modified to express the corresponding murine receptor. The cellular assay was used to screen 26,048 compounds selected for maximum diversity from a 200,640-compound in-house library. This yielded z' values greater than 0.8 with a hit rate of 3.3% and a confirmation rate of 50%. We selected 93 hits and enriched the collection with 279 similar compounds from the in-house library to identify promising structural features. The most active compounds were validated using orthogonal assay formats. The similarity of the compound profiles across the different platforms demonstrated that the reported lentiviral assay system is a robust and versatile tool for the identification of novel HIV-1 inhibitors.

Cancer is a devastating disease characterized by uncontrolled and aggressive cell growth. Suicide gene therapy (SGT) facilitating induction of malignancy-specific cell death represents a novel therapeutic approach to treat cancer, which has been investigated in several cancer types with very promising results. In addition, SGT has been suggested as a safeguard in adoptive immunotherapy and regenerative-medicine settings. Generally, SGT consists of two steps-vector-mediated delivery of suicide genes into tumors and subsequent activation of the suicide mechanism, e.g., by administration of a specific prodrug. This chapter provides a framework of protocols for basic and translational research using the Herpes-simplex-virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system, the most widely used suicide gene approach. The protocols provide standard guidelines for the preparation of high-titer third-generation lentiviral vectors encoding a genetically improved HSV-TK version known as TK.007 and its application in in vitro and in vivo treatment setups.

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: Mesenchymal stem (or stromal) cells (MSCs) have been used in more than 400 clinical trials for the treatment of various diseases. The clinical benefit and reproducibility of results, however, remain extremely variable. During the in vitro expansion phase, which is necessary to achieve clinically relevant cell numbers, MSCs show signs of aging accompanied by different contributions of single clones to the mass culture. Here we used multicolor lentiviral barcode labeling to follow the clonal dynamics during in vitro MSC expansion from whole umbilical cord pieces (UCPs). The clonal composition was analyzed by a combination of flow cytometry, fluorescence microscopy, and deep sequencing. Starting with highly complex cell populations, we observed a massive reduction in diversity, transiently dominating populations, and a selection of single clones over time. Importantly, the first wave of clonal constriction already occurred in the early passages during MSC expansion. Consecutive MSC cultures from the same UCP implied the existence of more primitive, MSC culture-initiating cells. Our results show that microscopically homogenous MSC mass cultures consist of many subpopulations, which undergo clonal selection and have different capabilities. Among other factors, the clonal composition of the graft might have an impact on the functional properties of MSCs in experimental and clinical settings., Significance: Mesenchymal stem cells (MSCs) can easily be obtained from various adult or embryonal tissues and are frequently used in clinical trials. For their clinical application, MSCs have to be expanded in vitro. This unavoidable step influences the features of MSCs, so that clinical benefit and experimental results are often highly variable. Despite a homogenous appearance under the microscope, MSC cultures undergo massive clonal selection over time. Multicolor fluorescence labeling and deep sequencing were used to demonstrate the dynamic clonal composition of MSC cultures, which might ultimately explain the variable clinical performance of the cells., (©AlphaMed Press.)

In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease.

TAL-effector nucleases (TALENs) are attractive tools for sequence-specific genome modifications, but their delivery still remains problematic. It is well known that the presence of multiple sequence repeats in TALEN genes hampers the use of lentiviral vectors. We report that lentiviral vectors readily package full-length vector mRNAs encoding TALENs, but recombination during reverse transcription prevents successful delivery. We reasoned that preventing reverse transcription of lentiviral-vector RNA would allow transfer of TALENs as mRNA. We demonstrate that lentiviral particles containing genetically inactivated reverse transcriptase (RT) mediated efficient transduction of cultured cells and supported transient transgene expression. For proof-of-principle, we transferred CCR5- and TCR-specific TALEN pairs for efficient targeted genome editing and abrogated expression for each of the receptor proteins in different cell lines. Combining the high specificity of TALENs with efficient lentiviral gene delivery should advance genome editing in vitro and potentially in vivo, and RT-deficient lentiviral vectors may be useful for transient expression of various other genes-of-interest.

Primary human B cells are an attractive target for gene-therapeutic applications, but have been found to be relatively resistant toward transduction with lentiviral vectors (LVVs), even though a number of different envelope pseudotypes were tested. Moreover, low transgene expression in primary human B cells has impeded the use of LVVs for this target cell. We investigated the transduction potential of gibbon-ape leukemia virus (GALV) Env-pseudotyped LVVs for primary human B cells. By establishing optimized transduction kinetics and multiplicities of infection, we were able to regularly obtain transduction efficiencies of more than 50% in CD40L-activated B cells. Noteworthy, with the use of GALV-pseudotyped LVVs we could achieve a more than 10-fold higher yield of transduced activated B cells in direct comparison with LVVs pseudotyped with measles virus glycoproteins. Phenotyping of transduced primary B cells revealed a majority of memory B cells, a long-lived phenotype, presumed to be well suited for enduring therapeutic interventions. Finally, by combining the enhancer (Eμ) and the matrix/scaffold-attachment regions (MARs) of the human immunoglobulin heavy chain with the promoter of spleen focus-forming virus (SFFV) we aimed at generating a novel LVV particularly suitable for B cell transgenesis. We show that the optimized vector facilitated significantly higher transgene expression in various B cell lines and, more importantly, primary human B cells (mean factor of three). In summary, we have established a novel protocol for the efficient lentiviral transduction of primary human B cells and have improved transgene expression in B cells by a specific vector modification.

Conditional elimination of infused gene-modified alloreactive T cells, using suicide gene activation, has been shown to be an efficient strategy to abrogate severe graft-versus-host disease (GvHD) in the context of adoptive immunotherapy. To overcome shortcomings of the most widely used suicide gene, wild-type (splice-corrected) herpes simplex virus thymidine kinase (scHSVtk), we generated two new variants: the codon-optimized coHSVtk and, by introducing an additional mutation (A168H), the novel TK.007. We transduced human hematopoietic cell lines and primary T cells with retroviral "sort-suicide vectors" encoding combinations of selection markers (tCD34 and OuaSelect) with one of three HSVtk variants. In vitro we observed higher expression levels and sustained long-term expression of TK.007, indicating lower nonspecific toxicity. Also, we noted significantly improved kinetics of ganciclovir (GCV)-mediated killing for TK.007-transduced cells. In an experimental (murine) allogeneic transplantation model, TK.007-transduced T cells mediated severe GvHD, which was readily abrogated by application of GCV (10 mg/kg). Last, we established a modified allotransplantation model that allowed quantitative comparison of the in vivo activities of TK.007 versus scHSVtk. We found that TK.007 mediates both significantly faster and higher absolute killing at low GCV concentrations (10 and 25 mg/kg). In summary, we demonstrate that the novel TK.007 suicide gene combines better killing performance with reduced nonspecific toxicity (as compared with the frequently used splice-corrected wild-type scHSVtk gene), thus representing a promising alternative for suicide gene therapy.

Stable genetic modification of stem cells holds great promise for gene therapy and marking, but commonly used gamma-retroviral vectors were found to influence growth/survival characteristics of hematopoietic stem cells (HSCs) by insertional mutagenesis. In this article, we show that promoter-deprived gamma-retroviral self-inactivating (pd-SIN) vectors allow stable genetic marking of serially reconstituting murine HSC. In contrast to findings with gamma-retroviral long terminal repeat (LTR) vectors, serial transplantation of pd-SIN-marked HSC in a sensitive mouse model was apparently not associated with induced clonal imbalance of gene-marked HSC. Furthermore, insertions of pd-SIN into protooncogenes, growth-promoting and signaling genes occurred significantly less frequent than in control experiments with LTR vectors. Also, transcriptional dysregulation of neighboring genes potentially caused by the pd-SIN insertion was rarely seen and comparatively weak. The integration pattern of promotor-deprived SIN vectors in reconstituting HSC seems to depend on the transcriptional activity of the respective gene loci reflecting the picture described for LTR vectors. In conclusion, our data strongly support the use of SIN vectors for gene-marking studies and suggest an increased therapeutic index for vectors lacking enhancers active in HSC.

Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

Evidence from model organisms and clinical trials reveals that the random insertion of retrovirus-based vectors in the genome of long-term repopulating hematopoietic cells may increase self-renewal or initiate malignant transformation. Clonal dominance of nonmalignant cells is a particularly interesting phenotype as it may be caused by the dysregulation of genes that affect self-renewal and competitive fitness. We have accumulated 280 retrovirus vector insertion sites (RVISs) from murine long-term studies resulting in benign or malignant clonal dominance. RVISs (22.5%) are located in or near (up to 100 kb [kilobase]) to known proto-oncogenes, 49.6% in signaling genes, and 27.9% in other or unknown genes. The resulting insertional dominance database (IDDb) shows substantial overlaps with the transcriptome of hematopoietic stem/progenitor cells and the retrovirus-tagged cancer gene database (RTCGD). RVISs preferentially marked genes with high expression in hematopoietic stem/progenitor cells, and Gene Ontology revealed an overrepresentation of genes associated with cell-cycle control, apoptosis signaling, and transcriptional regulation, including major "stemness" pathways. The IDDb forms a powerful resource for the identification of genes that stimulate or transform hematopoietic stem/progenitor cells and is an important reference for vector biosafety studies in human gene therapy.

Increasing evidence reveals that random insertion of gene transfer vectors into the genome of repopulating hematopoietic cells may alter their fate in vivo. Although most insertional mutations are expected to have few if any consequences for cellular survival, clonal dominance caused by retroviral vector insertions in (or in the vicinity of) proto-oncogenes or other signaling genes has been described for both normal and malignant hematopoiesis. Important insights into these side effects were initially obtained in murine models. Results from ongoing clinical studies have revealed that similar adverse events may also occur in human gene therapy. However, it remains unknown to what extent the outcome of insertional mutagenesis induced by gene vectors is related to (1) the architecture and type of vector used, (2) intrinsic properties of the target cell, and (3) extrinsic and potentially disease-specific factors influencing clonal competition in vivo. This review discusses reports addressing these questions, underlining the need for models that demonstrate and quantify the functional consequences of insertional mutagenesis. Improving vector design appears to be the most straightforward approach to increase safety, provided all relevant cofactors are considered.

Gene marking with replication-defective retroviral vectors has been used for more than 20 years to track the in vivo fate of cell clones. We demonstrate that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis. All 29 insertions recovered from clones dominating in serially transplanted recipients affected loci with an established or potential role in the self-renewal or survival of hematopoietic stem cells. Transcriptional dysregulation occurred in all 12 insertion sites analyzed. These findings have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover.

Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.

Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.

Objective: Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods., Materials and Methods: HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice., Results: After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7%+/-21.7%, n=23) and 30 to 95% (69.8%+/-20.4%, n=17), respectively. Even after one round of infection, gene transfer was efficient (31.2%+/-15.1%, n=12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells., Conclusion: The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.

Highly conserved enhancer sequences located in the upstream part of the long terminal repeat (LTR) of murine leukemia retroviruses (MLV) were reported to compromise viral gene expression in multipotent embryonic cells in vitro and to reduce the likelihood for maintenance of retroviral gene expression in hematopoietic cells in vivo. We show that deletion of these sequences (nucleotides +37 to +95) attenuates rather than increases the transcriptional activity of retroviral vectors in hematopoietic cells almost independently of the developmental lineage (erythroid, myeloid, or lymphoid). Expression rates of modified vectors were reduced by as much as 34-65%, although the strong enhancer array located in the direct repeat of the LTR was preserved. Sequence analysis and electrophoretic mobility shift assays revealed the presence of a highly conserved binding site for NFAT (nuclear factor of activated T cells) proteins that immediately neighbors a known binding site for the transcription factor Yin-Yang1 (YY1) [corrected]. Specific inactivation of the NFAT site reduced transgene expression in all cell types investigated and had a similar effect as the destruction of a neighboring SP1 motif. Combined destruction of individual motifs for NFAT, SP1, and E twenty-six transcription factors (ETS) resulted in a severe attenuation (by 40-60%) of the retroviral enhancer. These results provide novel clues for the manipulation of retrovirus replication and vector tropism.

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application. [ABSTRACT FROM AUTHOR]

Monitoring the fate of individual cell clones is an important task to better understand normal tissue regeneration, for example after hematopoietic stem cell (HSC) transplantation, but also cancerogenesis. Based on their integration into the host cell's genome, retroviral vectors are commonly used to stably mark target cells and their progeny. The development of genetic barcoding techniques has opened new possibilities to determine clonal composition and dynamics in great detail. A modular genetic barcode was recently introduced consisting of 32 variable positions (BC32) with a customized backbone, and its advantages were demonstrated with regard to barcode calling and quantification. The study presented applied the BC32 system in a complex in vivo situation, namely to analyze clonal reconstitution dynamics for HSC grafts consisting of up to three cell populations with distinguishable barcodes using different alpha- and lentiviral vectors. In a competitive transplantation setup, it was possible to follow the differently marked cell populations within individual animals. This enabled the clonal contribution of the different BC32 constructs during reconstitution and long-term hematopoiesis in the peripheral blood and the spatial distribution in bone marrow and spleen to be identified. Thus, it was demonstrated that the system allows the output of individually marked cells to be tracked in vivo and their influence on clonal dynamics to be analyzed. Successful application of the BC32 system in a complex, competitive in vivo situation provided proof-of-principle that its high complexity and the large Hamming distance between individual barcodes, combined with the easy customization, facilitate efficient and precise quantification, even without prior knowledge of individual barcode sequences. Importantly, simultaneous high-sensitivity analyses of different cell populations in single animals may significantly reduce numbers of animals required to investigate specific scientific questions in accordance with RRR principles. It is concluded that this BC32 system will be excellently suited for various research applications in regenerative medicine and cancer biology. [ABSTRACT FROM AUTHOR]

Besides its broad application in research and biotechnology, genome editing (GE) has great potential for clinical gene therapy, but delivery of GE tools remains a bottleneck. Whereas significant progress has been made in ex vivo GE delivery (e.g., by electroporation), establishment of efficient and safe in vivo delivery systems is still a challenge. Above and beyond standard vector requirements (safety, minimal/absent toxicity and immunogenicity, sufficient packaging capacity, targeting, straight and low-cost large-scale good manufacturing practice (GMP) production), GE delivery systems ideally use a hit-and-run principle to minimize off-targets as well as display of immunogenic peptides. Since currently used viral vectors do not fulfil all of these requirements, the broad variety of non-viral delivery platforms represents a promising alternative. This review provides a comprehensive analysis of the most relevant aspects of non-viral physical and chemical delivery methods in non-clinical studies and clinical trials, ranging from classic electroporation to advanced drug carriers that can transport GE tools in form of plasmid DNAs (pDNAs), mRNAs, and ribonucleoproteins (RNPs). For comparison, advantages and shortcomings of viral delivery systems are shortly discussed. In summary, we review various delivery approaches and discuss the future perspectives to use drug carriers for in vivo GE in clinical trials. Image 1 [ABSTRACT FROM AUTHOR]