Retroviral particle-mediated mRNA transfer

[no abstract

Gammaretroviral Vectors: Biology, Technology and Application

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

Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery

Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells

An apparatus for in-vacuum loading of nanoparticles into an optical trap

We describe the design, construction, and operation of an apparatus utilizing a piezoelectric transducer for in-vacuum loading of nanoparticles into an optical trap for use in levitated optomechanics experiments. In contrast to commonly used nebulizer-based trap-loading methods which generate aerosolized liquid droplets containing nanoparticles, the method produces dry aerosols of both spherical and high-aspect ratio particles ranging in size by approximately two orders of mangitude. The device has been shown to generate accelerations of order $10^7$ $g$, which is sufficient to overcome stiction forces between glass nanoparticles and a glass substrate for particles as small as $170$ nm diameter. Particles with sizes ranging from $170$ nm to $\sim 10$ $\mu$m have been successfully loaded into optical traps at pressures ranging from $1$ bar to $0.6$ mbar. We report the velocity distribution of the particles launched from the substrate and our results indicate promise for direct loading into ultra-high-vacuum with sufficient laser feedback cooling. This loading technique could be useful for the development of compact fieldable sensors based on optically levitated nanoparticles as well as matter-wave interference experiments with ultra-cold nano-objects which rely on multiple repeated free-fall measurements and thus require rapid trap re-loading in high vacuum conditions.Comment: 9 pages, 10 figure

The guanine-nucleotide exchange factor CalDAG GEFI fine-tunes functional properties of regulatory T cells

Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3(+) regulatory and Foxp3(-) conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI(-/-) mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI(-/-) Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI(+/+) Tregs. Interestingly, when tested in vivo, CalDAG GEFI(-/-) Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI(+/+) Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI(-/-) Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential

Genetic Deletion of SEPT7 Reveals a Cell Type-Specific Role of Septins in Microtubule Destabilization for the Completion of Cytokinesis

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wildtype genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model

Cellular Restriction of Retrovirus Particle-Mediated mRNA Transfer▿

Analyzing cellular restriction mechanisms provides insight into viral replication strategies, identifies targets for antiviral drug design, and is crucial for the development of novel tools for experimental or therapeutic delivery of genetic information. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription mediate a transient expression of any sequence which replaces the gag-pol transcription unit, a process we call retrovirus particle-mediated mRNA transfer (RMT). Here, we further examined the mechanism of RMT by testing its sensitivity to cellular restriction factors and short hairpin RNAs (shRNAs). We found that both human TRIM5α and, to a lesser extent, Fv1 effectively restrict RMT if the RNA is delivered by a restriction-sensitive capsid. While TRIM5α restriction of RMT led to reduced levels of retroviral mRNA in target cells, restriction by Fv1 did not. Treatment with the proteasome inhibitor MG132 partially relieved TRIM5α-mediated restriction of RMT. Finally, cells expressing shRNAs specifically targeting the retroviral mRNA inhibited RMT particles, but not reverse-transcribing particles. Retroviral mRNA may thus serve as a translation template if not used as a template for reverse transcription. Our data imply that retroviral nucleic acids become accessible to host factors, including ribosomes, as a result of particle remodeling during cytoplasmic trafficking