826. Transduction of Human Hematopoietic Stem Cells by RD114-TR-Pseudotyped Lentiviral Vectors

HIV-1-derived lentiviral vectors are efficiently pseudotyped by a chimeric envelope (RD114-TR) encoding the extracellular and transmembrane domains of the FLV RD114 glycoprotein fused to cytoplasmic tail (TR) of the MLV 4070A amphotropic glycoprotein. RD114-TR pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are of particular interest for both ex vivo and in vivo gene therapy applications. We carried out a comparative analysis of VSV-G and RD114-TR-pseudotyped lentiviral vectors in transducing human cord blood-derived CD34+ hematopoietic stem/progenitor cells. Transduction efficiency was comparatively analysed in CD34+ cells in liquid culture, in the progeny of CD34+ clonogenic progenitors in semi-solid culture, and in the progeny of CD34+ repopulating stem cells after xeno-transplantation in NOD-SCID mice. In all cases, RD114-TR-pseudotyped vectors transduced hematopoietic cells at lower m.o.i., resulting in lower toxicity and more efficient stable transduction at comparable vector copy number per genome. Potential changes in CD34+ cells transcription profile and phenotype upon transduction with RD114-TR or VSV-G-pseudotyped vectors was investigated by Affymetrix Gene Chips microarray analysis. We found no significant difference in gene expression patterns between mock-RD114-TR and VSV-G-transduced cells. Our study show that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors

Spin-orbit readout using thin films of topological insulator Sb2Te3 deposited by industrial magnetron sputtering

Driving a spin-logic circuit requires the production of a large output signal by spin-charge interconversion in spin-orbit readout devices. This should be possible by using topological insulators, which are known for their high spin-charge interconversion efficiency. However, high-quality topological insulators have so far only been obtained on a small scale, or with large scale deposition techniques which are not compatible with conventional industrial deposition processes. The nanopatterning and electrical spin injection into these materials has also proven difficult due to their fragile structure and low spin conductance. We present the fabrication of a spin-orbit readout device from the topological insulator Sb2Te3 deposited by large-scale industrial magnetron sputtering on SiO2. Despite a modification of the Sb2Te3 layer structural properties during the device nanofabrication, we measured a sizeable output voltage that can be unambiguously ascribed to a spin-charge interconversion process

Functional and Biochemical Characterization of Hepatitis C Virus (HCV) Particles Produced in a Humanized Liver Mouse Model

Lipoprotein components are crucial factors for hepatitis C virus (HCV) assembly and entry. As hepatoma cells producing cell culture-derived HCV (HCVcc) particles are impaired in some aspects of lipoprotein metabolism, it is of upmost interest to biochemically and functionally characterize the in vivo produced viral particles, particularly regarding how lipoprotein components modulate HCV entry by lipid transfer receptors such as scavenger receptor BI (SR-BI). Sera from HCVcc-infected liver humanized FRG mice were separated by density gradients. Viral subpopulations, termed HCVfrg particles, were characterized for their physical properties, apolipoprotein association, and infectivity. We demonstrate that, in contrast to the widely spread distribution of apolipoproteins across the different HCVcc subpopulations, the most infectious HCVfrg particles are highly enriched in apoE, suggesting that such apolipoprotein enrichment plays a role for entry of in vivo derived infectious particles likely via usage of apolipoprotein receptors. Consistent with this salient feature, we further reveal previously undefined functionalities of SR-BI in promoting entry of in vivo produced HCV. First, unlike HCVcc, SR-BI is a particularly limiting factor for entry of HCVfrg subpopulations of very low density. Second, HCVfrg entry involves SR-BI lipid transfer activity but not its capacity to bind to the viral glycoprotein E2. In conclusion, we demonstrate that composition and biophysical properties of the different subpopulations of in vivo produced HCVfrg particles modulate their levels of infectivity and receptor usage, hereby featuring divergences with in vitro produced HCVcc particles and highlighting the powerfulness of this in vivo model for the functional study of the interplay between HCV and liver components

Ultrafast spin-currents and charge conversion at \u3ci\u3e3d-5d\u3c/i\u3e interfaces probed by time-domain terahertz spectroscopy

Spintronic structures are extensively investigated for their spin-orbit torque properties, required for magnetic commutation functionalities. Current progress in these materials is dependent on the interface engineering for the optimization of spin transmission. Here, we advance the analysis of ultrafast spin-charge conversion phenomena at ferromagnetic-Transition metal interfaces due to their inverse spin-Hall effect properties. In particular, the intrinsic inverse spin-Hall effect of Pt-based systems and extrinsic inverse spin-Hall effect of Au:W and Au:Ta in NiFe/Au:(W,Ta) bilayers are investigated. The spin-charge conversion is probed by complementary techniques-ultrafast THz time-domain spectroscopy in the dynamic regime for THz pulse emission and ferromagnetic resonance spin-pumping measurements in the GHz regime in the steady state-to determine the role played by the material properties, resistivities, spin transmission at metallic interfaces, and spin-flip rates. These measurements show the correspondence between the THz time-domain spectroscopy and ferromagnetic spin-pumping for the different set of samples in term of the spin mixing conductance. The latter quantity is a critical parameter, determining the strength of the THz emission from spintronic interfaces. This is further supported by ab initio calculations, simulations, and analysis of the spin-diffusion and spin-relaxation of carriers within the multilayers in the time domain, permitting one to determine the main trends and the role of spin transmission at interfaces. This work illustrates that time-domain spectroscopy for spin-based THz emission is a powerful technique to probe spin-dynamics at active spintronic interfaces and to extract key material properties for spin-charge conversion

T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates.

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.This work was supported by European Union contract QLK2-CT-1999- 00356, by the Biomedical Primate Research Centre, The Netherlands, and by the Swedish Research Council. We are grateful to Alexander van den Berg for technical assistance with the ICS, to our colleagues from Animal Science Department for technical assistance and expert care of the macaques, to the participants of the European HCVacc Cluster who provided help and support, and to Thomas Darton (Oxford Vaccine Group, UK) for input and advice on the manuscript. Christine Rollier is an Oxford Martin fellow and a Jenner Insitute Investigator.This is the author accepted manuscript. The final version is available from Nature Publishing Group at https://doi.org/10.1038/gt.2016.55

Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology–in particular, in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies

Computing supersingular isogenies on Kummer surfaces

We apply Scholten\u27s construction to give explicit isogenies between the Weil restriction of supersingular Montgomery curves with full rational 2-torsion over $GF(p^2)$ and corresponding abelian surfaces over $GF(p)$. Subsequently, we show that isogeny-based public key cryptography can exploit the fast Kummer surface arithmetic that arises from the theory of theta functions. In particular, we show that chains of 2-isogenies between elliptic curves can instead be computed as chains of Richelot (2,2)-isogenies between Kummer surfaces. This gives rise to new possibilities for efficient supersingular isogeny-based cryptography

Selective gene silencing by viral delivery of short hairpin RNA

RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells

The human cytomegalovirus ul11 protein interacts with the receptor tyrosine phosphatase cd45, resulting in functional paralysis of t cells

Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV