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Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and pr...

The COVID-19 pandemic has led to rising morbidity, mortality, and social and economic disruption, stressors which have impaired mental health.1 Evidence emerged of substantial increases in anxiety and depression in spring and summer, 2020.2-7 To understand the effects of the pandemic and inform public health initiatives, it is essential to track trends in mental health disorders, as well as use of and need for mental health services, and to identify demographic groups at highest risk. Analyzing cross-sectional samples of 1,483,378 US adults, we show that reports of anxiety and depression rose significantly from April to November, 2020 to rates six-times higher than early 2019 US norms. Use of prescription medication, counseling services, and unmet need for mental health services also rose significantly. Prevalence rates of mental health disorders were highest among young, less educated, single, female, Black, Hispanic, and other race/ethnicity adults, with age and education disparities growing over time. Hispanics, Blacks, other race/ethnicities, and less educated respondents also were significantly less likely to be receiving medication or counseling and reported higher unmet needs for services. Together, disparities in estimates of mental health disorders and mental health treatment indicate a striking disequilibrium between the potential need for and the use of mental health services during the COVID-19 pandemic. Rising mental health challenges are being born largely by young, less advantaged people of color and women, with the potential for expanded interruptions to optimal functioning and repercussions for social and economic recovery from COVID-19.

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS)

The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Because current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout. By redirecting the gammaretroviral packaging machinery, we transiently delivered Streptococcus pyogenes Cas9 (SpCas9) mRNA and single-guide RNA transcripts into various (including primary) cell types. Spatiotemporal co-delivery of CRISPR/Cas9 components resulted in efficient disruption of a surrogate reporter gene, as well as functional knockout of endogenous human genes CXCR4 and TP53. Although acting in a hit-and-run fashion, knockout efficiencies of our transient particles corresponded to 52%–80% of those obtained from constitutively active integrating vectors. Stable SpCas9 overexpression at high doses in murine NIH3T3 cells caused a substantial G0/G1 arrest accompanied by reduced cell growth and metabolic activity, which was prevented by transient SpCas9 transfer. In summary, the non-integrating retrovirus-based vector particles introduced here allow efficient and dose-controlled delivery of CRISPR/Cas9 components into target cells. Keywords: CRISPR/Cas9 all-in-one particle, transient delivery, cytotoxicity, gammaretroviral MS2 chimera, targeted gene knockout, genome editing

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV., Author Summary Current antiretroviral combination therapy can efficiently suppress virus replication, but cannot eliminate HIV. Therefore, no cure for HIV exists. A main hurdle for virus eradication is seen in the existence of resting cells that contain integrated replication-competent, but temporarily silenced, HIV genomes. Therefore, the most direct approach to eliminating virus reservoirs is to remove HIV genomes from infected cells. As previous studies suggested, this may be achievable by Tre-recombinase, an engineered enzyme that can excise integrated HIV from host cell chromosomes. The present work analyzes the expression of Tre-recombinase in human cells and demonstrates highly accurate Tre activity in complete absence of Tre-related cytopathic effects. Furthermore, in vivo analysis of Tre-recombinase demonstrates highly significant antiviral effects of Tre in HIV-infected humanized mice. The presented data suggest that Tre-recombinase might become a valuable component of a future therapy that aims at virus eradication.

Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible 'all-in-one' lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16-24 h) and dose-dependent (>0.04 μg ml(-1) Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34(+) hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-β-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34(+)-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.

High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). The activity of the master regulator of this pathway, PTEN, is often impaired in T-ALL. However, experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is required for sustained mTOR activation, even in the absence of PTEN. We previously reported the expression of Neurotrophin receptor tyrosine kinases (TRKs) and their respective ligands in primary human leukemia samples. In the present study we aimed to dissect the downstream signaling cascades of TRK-induced T-ALL in a murine model and show that T-ALLs induced by deregulated receptor tyrosine kinase signaling acquire activating mutations in Notch1 and lose PTEN during clonal evolution. Some clones additionally lost one allele of the homeodomain transcription factor Cux1. All events independently led to a gradual hyperactivation of both mTORC1 and mTORC2 signaling. We dissected the role of the individual mTOR complexes by shRNA knockdown and found that the separate depletion of mTORC1 or mTORC2 reduced the growth of T-ALL blasts, but was not sufficient to induce apoptosis. In contrast, knockdown of the mTOR downstream effector eIF4E caused a striking cytotoxic effect, demonstrating a critical addiction to cap-dependent mRNA-translation. Although high mTORC2-AKT activation is commonly associated with drug-resistance, we demonstrate that T-ALL displaying a strong mTORC2-AKT activation were specifically susceptible to 4EGI-1, an inhibitor of the eIF4E-eIF4G interaction. To decipher the mechanism of 4EGI-1, we performed a genome-wide analysis of mRNAs that are translationally regulated by 4EGI-1 in T-ALL. 4EGI-1 effectively reduced the ribosomal occupancy of mRNAs that were strongly upregulated in T-ALL blasts compared with normal thymocytes including transcripts important for translation, mitochondria and cell cycle progression, such as cyclins and ribosomal proteins. These data suggest that disrupting the eIF4E-eIF4G interaction constitutes a promising therapy strategy in mTOR-deregulated T-cell leukemia.

Retroviral vectors are versatile gene transfer vehicles widely used in basic research and gene therapy. Mutation of retroviral integrase converts these vectors into transient, integration-deficient gene delivery vehicles associated with a high degree of biosafety. We explored the option to use integration-deficient retroviral vectors to achieve transient ectopic expression of transcription factors, which is considered an important tool for induced cell fate conversion. Stepwise optimization of the retroviral episome transfer as exemplified for the transcription factor Oct4 enabled to improve both expression magnitude and endurance. Long terminal repeat-driven γ-retroviral vectors were identified as the most suitable vector architecture. Episomal expression was enhanced by epigenetic modifiers, and Oct4 activity was increased following fusion to a minimal transactivation motif of herpes simplex virus VP16. Based on kinetic analyses, we identified optimal time intervals for repeated vector administration and established prolonged expression windows of choice. Providing proof-of-concept, episomal transfer of Oct4 was potent to mediate conversion of human fibroblasts stably expressing Klf4, Sox2 and c-Myc into induced pluripotent stem cells, which were mainly free of residual Oct4 vector integration. This study provides evidence for suitability of retroviral episome transfer of transcription factors for cell fate conversion, allowing the generation of distinct patient- or disease-specific cell types.

Site specific recombinases are frequently used as gene switches in transgenic animals where recombination is induced by drug treatment or by tissue specific recombinase expression. Alternatively, lentiviral gene transfer can be utilized for the genetic modification of a wide variety of cell types, albeit systems for tight control of transcriptional activity are scarce. Here, we combined lentiviral gene transfer and the development of a tightly drug-controlled FLP recombinase for the construction of “All-in-One” inducible gene expression systems. Tight control of FLP activity was achieved through N-terminal fusion with a FKBP12-derived conditional destruction domain and a C-terminal estrogen receptor binding domain making recombination dependent on the presence of Shield–1 and 4-hydroxytamoxifen. Exploiting the capacity of FLP to mediate excision and inversion, “All-in-One” lentiviral gene switch vector systems were generated where drug-induced recombination resulted in abrogation of FLP expression and subsequent overexpression or knockdown of the prototypical tumor suppressor phosphatase and tensin homolog PTEN. “All-in-One” vectors proved their functionality in a variety of hematopoietic cell lines, and primary murine bone marrow cells. Our new vector system thus combines the ease of lentiviral gene transfer and the power of site specific recombinases for analysis of gene function.

Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for “safe harbor” integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy.

Lentiviral (LV) vectors are promising tools for long-term genetic correction of hereditary diseases. In hematopoietic stem cell gene therapies adverse events in patients due to vector integration-associated genotoxicity have been observed. Only a few studies have explored the potential risks of LV gene therapy targeting the liver. To analyze hepatic genotoxicity in vivo, we transferred the fumarylacetoacetate hydrolase (FAH) gene by LV vectors into FAH(-/-) mice (n = 97) and performed serial hepatocyte transplantations (four generations). The integration profile (4,349 mapped insertions) of the LV vectors was assessed by ligation-mediated polymerase chain reaction and deep sequencing. We tested whether the polyclonality of vector insertions was maintained in serially transplanted mice, linked the integration sites to global hepatocyte gene expression, and investigated the effects of LV liver gene therapy on the survival of the animals. The lifespan of in vivo gene-corrected mice was increased compared to 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) control animals and unchanged in serially transplanted animals. The integration profile (4,349 mapped insertions) remained polyclonal through all mouse generations with only mild clonal expansion. Genes close to the integration sites of expanding clones may be associated with enhanced hepatocyte proliferation capacity. Conclusion: We did not find evidence for vector-induced tumors. LV hepatic gene therapy showed a favorable risk profile for stable and long-term therapeutic gene expression. Polyclonality of hepatocyte regeneration was maintained even in an environment of enforced proliferation. (HEPATOLOGY 2013)

Retroviral gene transfer has proven therapeutic potential in clinical gene therapy trials but may also cause abnormal cell growth via perturbation of gene expression in the locus surrounding the insertion site. By establishing clonal marks, retroviral insertions are also used to describe the regenerative potential of individual cells. Deep sequencing approaches have become the method of choice to study insertion profiles in preclinical models and clinical trials. We used a protocol combining ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing for insertion profiling and quantification in cells of various tissues transduced with various retroviral vectors. The presented method allows simultaneous analysis of a multitude of DNA-barcoded samples per pyrosequencing run, thereby allowing cost-effective insertion screening in studies with multiple samples. In addition, we investigated whether the number of pyrosequencing reads can be used to quantify clonal abundance. By comparing pyrosequencing reads against site-specific quantitative PCR and by performing spike-in experiments, we show that considerable variation exists in the quantification of insertion sites even when present in the same clone. Our results suggest that the protocol used here and similar approaches might misinterpret abundance clones defined by insertion sites, unless careful calibration measures are taken. The crucial variables causing this variation need to be defined and methodological improvements are required to establish pyrosequencing reads as a quantification measure in polyclonal situations.

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild-type and histone methyltransferase Suv39h1-deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc-overexpressing wild-type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end-to-end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc-transduced Suv39h1-deficient cells showed less critically short telomeres (P < 0.05) compared with Myc-transduced wild-type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc.

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into ‘safe harbor’ sites in the genomes of autologous, patient-derived iPS cells.

The genetic modification of lymphocytes is an important topic in the emerging field of gene therapy. Many clinical trials targeting immunodeficiency syndromes or cancer have shown therapeutic benefit; further applications address inflammatory and infectious disorders. Retroviral vector development requires a detailed understanding of the interactions with the host. Most researchers have used simple gammaretroviral vectors to modify lymphocytes, either directly or via hematopoietic stem and progenitor cells. Lentiviral, spumaviral (foamyviral) and alpharetroviral vectors were designed to reduce the necessity for cell stimulation and to utilize potentially safer integration properties. Novel surface modifications (pseudotyping) and transgenes, built using synthetic components, expand the retroviral toolbox, altogether promising increased specificity and potency. Product consistency will be an important criterion for routine clinical use.

Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with γ-retroviral vectors, due to their integration bias to transcription units in comparison to the γ-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design.

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

OBJECTIVES: Methods to expand hematopoietic stem cells (HSCs) ex vivo encompass an attractive approach that would substantially broaden the clinical applicability of HSCs derived from cord blood. Recently, members of the Angiopoietin-like (Angptl) family of growth factors were shown to expand both murine and human HSCs. Specifically, Angptl5 has been implicated in the expansion of human NOD-SCID-repopulating cells (SRCs) ex vivo. Here, we sought to evaluate the potential of additional Angptls to expand human SRCs from cord blood. Additionally, the purpose of this study was to evaluate the reproducibility of Angptl-mediated expansion of SRCs across independent experiments. METHODS: Human CD34(+) cells from cord blood were cultured in vitro for eleven or eight days in the presence or absence of Angptls. The reconstitution capacity of expanded cells was subsequently measured in vivo by transplantation into NOD-SCID or NSG mice, and compared to that of uncultured cells. RESULTS: We report here that Angptl4 functions to maintain SRC-activity of CD34(+) CB-derived cells ex vivo as assayed in NOD-SCID and NSG mice. However, all Angptls tested, including Angptl1, 4, and 5, were associated with variation between experiments. CONCLUSION: Our findings indicate that Angptl4 and Angptl5 can lead to increased engraftment capacity of SRCs, but more frequently these factors are associated with maintenance of SRC-activity during ex vivo culture. Thus, Angptl-mediated expansion of SRCs ex vivo is associated with more inter-experimental variation than previously thought. We conclude that Angptls would be useful in instances where there is a need to maintain HSCs ex vivo, such as during transduction for gene therapy applications.

The effective entry of retroviruses into target cells depends on the presence of viral envelope (Env) proteins and cognate cellular receptors, such as the murine cationic amino acid transporter-1 (mCAT-1) for the ecotropic murine leukemia virus (MLV-E). Here, we examined whether human cells internalize MLV-E or other retroviral pseudotypes irrespective of the presence of a specific receptor. Using fluorescently tagged Gag to monitor viral internalization, and treating cells with chloroquine or bafilomycin A1, we show that endocytosis is the main pathway for productive transduction with ecotropic particles, but endocytosis of retroviral particles itself does not depend on a suitable receptor or Env. Nonspecific endosomal uptake and lysosomal degradation occurred with all "illegitimate" envelope-receptor combinations tested: MLV particles pseudotyped with the ecotropic envelope or measles virus H and F proteins as well as "ecotropic" or "bald" HIV-1 particles. Kinetic studies in cell lines and primary human T lymphocytes showed the persistence of Gag-GFP signals for more than 10 days after exposure to retroviral vector particles, even in the absence of a suitable receptor. Further studies testing the Gag-mediated transfer of protein or retroviral mRNA revealed that nonspecific endocytosis prevented the release of functional particle-associated proteins and nucleic acids into the cytosol. We conclude that receptor-targeted retroviral particles are unlikely to escape nonspecific cellular uptake unless appropriate protective principles are discovered. Conversely, as lysosomal degradation was found to inactivate mRNA and proteins embedded into retroviral particles, receptor targeting is a useful strategy for both transient and permanent cell modification by retrovirus-like particles.

Retroviral vectors represent evolutionarily optimized gene delivery vehicles, which stably integrate their coding DNA into the host cell genome. In contrast to other gene delivery platforms, retroviral entry and integration are relatively efficient due to the utilization of cellular mechanisms for particle transport, DNA repair and gene expression, features that can be exploited for gene therapy and cell modification. Arresting the retroviral life cycle at specific steps, i.e. prior to reverse transcription or integration, allows for the utilization of intermediate structures (mRNA) or by-products (episomes) as tools for transient applications. However, it is often overlooked that retroviral particles are composed of up to 2500 Gag structural proteins, as well as further proteins involved in viral replication, all of which can be harnessed for the transfer of heterologous proteins into target cells. In this review, we describe the general biology of retroviruses and their derived vector systems, and then discuss the potential of engineering their protein components. We focus on lentiviral, gammaretroviral and alpharetroviral vector systems, and address current developments in the visualization of retrovirus-cell interactions (live cell imaging), and potential applications of engineered retroviral particles in biotechnology and biomedical research. Compared to conventional protein transduction techniques, we envisage protein-transducing retrovirus-like particles as a highly flexible platform for the efficient and cell-targeted delivery of designer proteins, even in combination with transduction of retroviral mRNA, episomal DNA or integrating DNA.

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

The transient delivery of gene products (RNA or proteins) is not a biotechnological invention but rather an evolutionarily conserved process underlying and regulating a variety of biological functions. On the basis of insights into the underlying mechanisms, several viral and cell-based approaches have been developed for the delivery of RNA or proteins. Prominent applications include the induction of major biological or therapeutic effects on the basis of "hit-and-run" mechanisms, such as vaccination, cell fate modification (reprogramming, differentiation), control of cell trafficking, enhancement of cell regeneration, and genome engineering using sequence-specific recombinases or nucleases. Ideally, procedures for delivery of RNA or proteins should be targeted to specific cells, overcome biophysical hurdles without harming cellular integrity, circumvent the various alarm signals of the innate immune system, allow dose-controlled delivery of functional biomacromolecules, and avoid the induction of an adaptive immune response. Here we review the current state of approaches for the delivery of mRNA and proteins with a focus on RNA viruses, virus-like particles including retrovirus particle-mediated transfer of mRNA or proteins, extracellular vesicles, and cell-penetrating peptides. The basic concepts and recent advances are put into perspective in the context of potential limitations of the technologies and strategies to overcome cellular barriers and defense mechanisms.

The therapeutic use of procedures for genetic stem cell modification is limited by potential adverse events related to uncontrolled mutagenesis. Prominent findings have been made in hematopoietic gene therapy, demonstrating the risk of clonal, potentially malignant outgrowth on the basis of mutations acquired during or after therapeutic genome modification. The incidence and the growth rate of insertional mutants have been linked to the “stemness” of the target cells and vector-related features such as the integration pattern, the architecture, and the exact content of transgene cassettes. Milieu factors supporting the survival and expansion of mutants may eventually allow oncogenic progression. Similar concerns apply for medicinal products based on pluripotent stem cells. Focusing on the genetic stress induced by insertional mutagenesis and culture adaptation, we propose four conclusions. (a) Mutations occurring in the production of stem cell-based medicines may be unavoidable and need to be classified according to their risk to trigger the formation of clones that are sufficiently long-lived and mitotically active to acquire secondary transforming mutations. (b) The development of rational prevention strategies depends upon the identification of the specific mutations forming such “dominant clones” (which can also be addressed as cancer stem cell precursors) and a better knowledge of the mechanisms underlying their creation, expansion, and homeostatic control. (c) Quantitative assay systems are required to assess the practical value of preventive actions. (d) Improved approaches for the genetic modification of stem cells can address all critical steps in the origin and growth control of mutants.

Gene therapy is a promising therapeutic approach to treat primary immunodeficiencies. Indeed, the clinical trial for the Wiskott–Aldrich Syndrome (WAS) that is currently ongoing at the Hannover Medical School (Germany) has recently reported the correction of all affected cell lineages of the hematopoietic system in the first treated patients. However, an extensive study of the clonal inventory of those patients reveals that LMO2, CCND2 and MDS1/EVI1 were preferentially prevalent. Moreover, a first leukemia case was observed in this study, thus reinforcing the need of developing safer vectors for gene transfer into HSC in general. Here we present a novel self-inactivating (SIN) vector for the gene therapy of WAS that combines improved safety features. We used the elongation factor 1 alpha (EFS) promoter, which has been extensively evaluated in terms of safety profile, to drive a codon-optimized human WASP cDNA. To test vector performance in a more clinically relevant setting, we transduced murine HSPC as well as human CD34+ cells and also analyzed vector efficacy in their differentiated myeloid progeny. Our results show that our novel vector generates comparable WAS protein levels and is as effective as the clinically used LTR-driven vector. Therefore, the described SIN vectors appear to be good candidates for potential use in a safer new gene therapy protocol for WAS, with decreased risk of insertional mutagenesis. Fil: Avedillo Diez, Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Hannover Medical School; Alemania Fil: Zychlinski, Daniela. Hannover Medical School; Alemania Fil: Coci, Emanuele G.. Hannover Medical School; Alemania Fil: Galla, Melanie. Hannover Medical School; Alemania Fil: Modlich, Ute. Hannover Medical School; Alemania Fil: Dewey, Ricardo. Hannover Medical School; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Schwarzer, Adrian. Hannover Medical School; Alemania Fil: Maetzig, Tobias. Hannover Medical School; Alemania Fil: Mpofu, Nonsikelelo. Hannover Medical School; Alemania Fil: Jaeckel, Elmar. Hannover Medical School; Alemania Fil: Boztug, Kaan. Hannover Medical School; Alemania Fil: Baum, Christopher. Hannover Medical School; Alemania Fil: Klein, Christoph. Hannover Medical School; Alemania Fil: Schambach, Axel. Hannover Medical School; Alemania

Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with ∼30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (

Retroviruses are evolutionary optimized gene carriers that have naturally adapted to their hosts to efficiently deliver their nucleic acids into the target cell chromatin, thereby overcoming natural cellular barriers. Here we will review—starting with a deeper look into retroviral biology—how Murine Leukemia Virus (MLV), a simple gammaretrovirus, can be converted into an efficient vehicle of genetic therapeutics. Furthermore, we will describe how more rational vector backbones can be designed and how these so-called self-inactivating vectors can be pseudotyped and produced. Finally, we will provide an overview on existing clinical trials and how biosafety can be improved.

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Insertionsmutagenese beschreibt einen Formenkreis genotoxischer Effekte der Integration von Transgenen im zellularen Genom. Gefurchtet ist insbesondere die Aktivierung zellularer Proto-Onkogene durch transkriptionelle Deregulation oder strukturelle Anderung der kodierten Proteine. Klinische Konsequenzen mit Induktion schwerer Nebenwirkungen hat die Insertionsmutagenese bislang nur nach der genetischen Modifikation von Blutstamm- und Vorlauferzellen gezeigt. Als wesentliche Einflussgrosen wurden das Integrationsmuster der Genvektoren, die Art des Transgen-Promotors, die Art der Zielzelle und die Selektionsbedingungen im Organismus identifiziert. Entsprechend leiten sich rationale Ansatze fur eine wirksame Pravention dieses grundlegenden Risikos der Gentherapie ab. Die Weiterentwicklung der Genvektoren, eine proaktive, hypothesengetriebene Toxikologie und risikoadaptierte klinische Studien wirken zusammen, um die Gefahren der Insertionsmutagenese zu bannen und das Potenzial der Gentherapie mit der gebotenen Sicherheit zu erschliesen.

Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in “expanded” groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.

The regulated expression of therapeutic genes may become crucial in gene therapy when their constitutive expression interferes with cell fate in vivo. The efficient regulation of transgene expression requires tightly controlled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its application requires the introduction of two components into the target cell genome: the tet-responsive transactivator and the regulated expression cassette. In order to facilitate the usage of the tet-system for approaches in gene therapy, both components have to be transferred by a single vector, thus eliminating the preselection of transactivator positive cells. Published "all-in-one" vectors for regulated transgene expression display a relatively low signal-to-noise ratio, resulting in regulatory windows of around 500-fold even in selected clones. In this study, we show that a modified vector architecture combined with the introduction of new tet-responsive promoters, Ptet, improved the dynamic range of such all-in-one vectors to levels up to 14,000-fold for viral and 25,000-fold for nonviral transfer vectors in nonclonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improved regulation was the result of a strong reduction of background expression in the off-state, even if cells were transduced at high multiplicity of infection, while induction remained at high levels. In addition, the results indicated that successful regulation of gene expression in different target cells depended on vector architecture as well as the choice of the Ptet-promoter.

The 17th Annual Meeting of the German Society for Gene Therapy was held at the Chemistry and Pharmacy Campus of the University of Munich in conjunction with and supported by the British Society for Gene Therapy, the Viral Vectors Study Group of the German Society for Virology, the Research Priority Program SPP1230, the Nanosystems Initiative Munich and the Helmholtz Center Munich. The German Research Foundation provided financial support for the invited international speakers. In addition to 25 invited lectures, 21 oral presentations were selected out of more than 100 submitted abstracts. State-of-the-art advances in the field of gene therapy were presented, a field that has considerably evolved within recent years. More than 200 researchers from Germany and other European countries, as well as the USA, Canada and Japan attended the meeting. Prior to the official meeting, a public day was organized, in which the interested public could participate in talks and discussions concerning gene therapy issues. Furthermore, at the ‘kids workshop’ young scientists aged 8–10 years were discovering cellular and genetic mechanisms and the principles of gene therapy.

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor gamma gene (IL2RG) cDNA (co gamma c), regulated by its 1.1 kb promoter region (gamma cPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of IL2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and gamma cPr vectors. We conclude that SIN lentiviral gene therapy using co gamma c driven by the gamma cPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required. Received 23 February 2011; accepted 31 May 2011; published online 12 July 2011. doi:10.1038/mt.2011.127

The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.

Gene vectors with an untargeted insertion profile have been explored in preclinical models and clinical trials for the transfer of potentially therapeutic genetic information into somatic cells that have a high replicative potential. The gene-modified cell population can be viewed as a genetic mosaic whose complexity depends upon the target cell type, the number of transduced cells, the average number of insertions per cell, the genetic stability and composition of the transgene, and the integration pattern of the vector. Selection by the environment encountered in the patient may support the preferential survival of clones with insertional deregulation of genes that are involved in the control of engraftment, proliferation or differentiation, in the worst case initiating oncogenic progression. Rapid scientific and technological progress has shed much light onto this dark side of untargeted vector integration. New approaches to unbiased and highly sensitive "integromics" promise a precise documentation of stable polyclonality, clonal fluctuation or clonal imbalance of gene-modified cell populations. Evidence has been obtained for a number of approaches to potentially reduce the genomic risk of gene therapy: targeting cells that lack sustained replicative potential, using vectors with a more neutral integration spectrum, reducing the number of vector copies per cell, designing gene expression cassettes that avoid long-distance enhancer interactions or fusion transcripts, and reducing, as far as possible, the risk of secondary mutations. The genomic risk of gene therapy can thus be prevented by the collective targeting of all contributing factors.

The generation of induced pluripotent stem cells (iPSCs) enabled by exogenous expression of the canonical Oct4, Sox2, Klf4 and c-Myc reprogramming factors has opened new ways to create patient- or disease-specific pluripotent cells. iPSCs represent an almost inexhaustible source of cells for targeted differentiation into somatic effector cells and hence are likely to be invaluable for therapeutic applications and disease-related research.After an introduction on the biology of reprogramming we cover emerging technological advances, including new reprogramming approaches, small-molecule compounds and tailored genetic modification, and give an outlook towards potential clinical applications of iPSCs.Although this field is progressing rapidly, reprogramming is still an inefficient process. The reader will learn about innovative tools to generate patient-specific iPSCs and how to modify these established lines in a safe way. Ideally, the disease-causing mutation is edited directly in the genome using novel technologies based on artificial nucleases, such as zinc-finger nucleases.Human iPSCs create fascinating options with regard to disease modeling, drug testing, developmental studies and therapeutic applications. However, important hurdles have to be taken and more efficient protocols to be established to achieve the ambitious goal of bringing iPSCs into clinical use.

Signaling of the thrombopoietin (THPO) receptor MPL is critical for the maintenance of hematopoietic stem cells (HSCs) and megakaryocytic differentiation. Inherited loss-of-function mutations of MPL cause severe thrombocytopenia and aplastic anemia, a syndrome called congenital amegakaryocytic thrombocytopenia (CAMT). With the aim to assess the toxicity of retroviral expression of Mpl as a basis for further development of a gene therapy for this disorder, we expressed Mpl in a murine bone marrow transplantation (BMT) model. Treated mice developed a profound yet transient elevation of multilineage hematopoiesis, which showed morphologic features of a chronic myeloproliferative disorder (CMPD) with progressive pancytopenia. Ten percent of mice (3/27) developed erythroleukemia, associated with insertional activation of Sfpi1 and Fli1. The majority of transplanted mice developed a progressive pancytopenia with histopathological features of a myelodysplastic syndrome (MDS)–like disorder. To avoid these adverse reactions, improved retroviral vectors were designed that mediate reduced and more physiological Mpl expression. Self-inactivating γ-retroviral vectors were constructed that expressed Mpl from the phosphoglycerate kinase (PGK) or the murine Mpl promoter. Mice that received BM cells expressing Mpl from the Mpl promoter were free of any previously observed adverse reactions.

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans . Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.

Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer–promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott–Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.

Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were approximately 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O(6)-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression.

Multiple types of regulation are used by cells and viruses to control alternative splicing. In murine leukemia virus, accessibility of the 5′ splice site (ss) is regulated by an upstream region, which can fold into a complex RNA stem–loop structure. The underlying sequence of the structure itself is negligible, since most of it could be functionally replaced by a simple heterologous RNA stem–loop preserving the wild-type splicing pattern. Increasing the RNA duplex formation between U1 snRNA and the 5′ss by a compensatory mutation in position +6 led to enhanced splicing. Interestingly, this mutation affects splicing only in the context of the secondary structure, arguing for a dynamic interplay between structure and primary 5′ss sequence. The reduced 5′ss accessibility could also be counteracted by recruiting a splicing enhancer domain via a modified MS2 phage coat protein to a single binding site at the tip of the simple RNA stem–loop. The mechanism of 5′ss attenuation was revealed using hyperstable U1 snRNA mutants, showing that restricted U1 snRNP access is the cause of retroviral alternative splicing.

In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient gamma-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of approximately 10(-4). Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk10(-6)). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity.

Die Promotion in der Humanmedizin und die weiterfuhrende wissenschaftliche Qualifikation nehmen im Reigen der Naturwissenschaften eine Sonderstellung ein. Wahrend die Promotion zum Titel des Dr. med. oftmals einer Initiation in das wissenschaftliche Arbeiten gleichkommt, findet sich fur die vertiefende wissenschaftliche Tatigkeit nach dem erfolgreichen Abschluss des humanmedizinischen Studiums eine grose Vielfalt an Gestaltungsformen. In den letzten Jahren haben – den Vorgaben des Wissenschaftsrats und der Deutschen Forschungsgemeinschaft folgend – vermehrt strukturierte Programme fur medizinische und naturwissenschaftliche Promotionen Einzug in die humanmedizinische Ausbildung gefunden. An der Medizinischen Hochschule Hannover werden solche Programme seit Jahren angeboten. Das StrucMed-Programm erhoht die Qualitat studiumbegleitender medizinischer Promotionen insbesondere im Bereich molekularbiologischer und tierexperimenteller Projekte. Die Hannover Biomedical Research School (HBRS) vereint mehrere Graduiertenprogramme fur naturwissenschaftliche Promotionen und bietet zusammen mit dem StrucMed-Programm eine Plattform fur eine international ausgerichtete wissenschaftliche Ausbildung der Absolventen lebenswissenschaftlicher Studiengange. Eine engmaschige Evaluation der Ausbildungsleistungen und Karrierepfade der Absolventen soll die evidenzbasierte Integration wissenschaftlicher Arbeit in einem leistungsorientierten klinischen Umfeld erlauben.

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-α (TNF-α) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-α, we studied Fancc−/− HSCs to determine the physiologic effects of HOXB4 on TNF-α sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-α of Fancc−/− HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc−/− but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-α receptors on Fancc−/− HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-α signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.

Cell membrane-anchored (ma) antiviral peptides derived from the C-terminal heptad repeat of the HIV-1 transmembrane glycoprotein gp41 (C-peptides) and expressed from retroviral vectors were shown to efficiently inhibit HIV-1 entry into target cells. Here, we analyzed the influence of the vector backbone, the scaffold modules that anchor the peptide to the membrane and the length of the C-peptide on expression level and antiviral activity. In general, antiviral activity was determined primarily by the density of the C-peptide on the cell surface. By influencing expression levels, the scaffold elements indirectly also determined antiviral activity. Additional direct effects of the scaffold on antiviral activity were minor. At comparable expression levels, the elongated C-peptide (maC46) was found to be more potent than the shorter maC36. On the basis of these findings, a dose-response assay was established that quantifies antiviral activity relative to the expression level of the antiviral gene product. Taken together, these data demonstrate the importance of analyzing the efficacy of therapeutic genes relative to the dose of the gene product.

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase–polymerase chain reaction (RT-PCR), we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB+ cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target.