Immunogenicity of targeted lentivectors

To increase the safety and possibly efficacy of HIV-1 derived lentivectors (LVs) as an anti-cancer vaccine, we recently developed the Nanobody (Nb) display technology to target LVs to antigen presenting cells (APCs). In this study, we extend these data with exclusive targeting of LVs to conventional dendritic cells (DCs), which are believed to be the main cross-presenting APCs for the induction of a TH1-conducted antitumor immune response. The immunogenicity of these DC-subtype targeted LVs was compared to that of broad tropism, general APC-targeted and non-infectious LVs. Intranodal immunization with ovalbumin encoding LVs induced proliferation of antigen specific CD4(+) T cells, irrespective of the LVs' targeting ability. However, the cytokine secretion profile of the restimulated CD4(+) T cells demonstrated that general APC targeting induced a similar TH1-profile as the broad tropism LVs while transduction of conventional DCs alone induced a similar and less potent TH1 profile as the non-infectious LVs. This observation contradicts the hypothesis that conventional DCs are the most important APCs and suggests that the activation of other APCs is also meaningful. Despite these differences, all targeted LVs were able to stimulate cytotoxic T lymphocytes, be it to a lesser extent than broad tropism LVs. Furthermore this induction was shown to be dependent on type I interferon for the targeted and non-infectious LVs, but not for broad tropism LVs. Finally we demonstrated that the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such deliver on their promise as safer and efficacious LV-based vaccines

Site Specific Knock-In Genome Editing in Human HSCs Using Baboon Envelope gp Pseudotypedviral Derived “Nanoblades” Loaded with Cas9/sgRNA Combined with Donor Encoding AAV-6

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. Here, we have designed ?Nanoblades?, a new technology that will deliver a genomic cleaving agent into cells. These are genetically modified Murine Leukemia Virus (MLV) or HIV derived virus like particle (VLP), in which the viral structural protein Gag has been fused to the Cas9. These VLPs are thus loaded with Cas9 protein together with the guide RNAs. Thus, nanoblades are devoid of any viral-derived genetic material. Highly efficient gene editing was obtained in cell lines, IPS cells and primary mouse and human cells (Mangeot et al. Nature Communication, 2019). However, their delivery into target cells can be technically challenging when working with primary immune cells. Now we showed that nanoblades were remarkably efficient for entry into human T, B and hematopoietic stem cells thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins. We were able to induce efficient, transient and very rapidlygenome-editing in human induced pluripotent stem cells reaching up to 70% in the empty spiracles homeobox 1 (EMX1) and muscular dystrophy (MD) gene locus. A brief nanoblade incubation of primary human T and B cells resulted in 40% and 20% editing of the Wiskott-Aldrich syndrome (WAS) gene locus, while hematopoietic stem cells treated for 18 h with nanoblades allowed 30-40% gene editing in the WAS gene locus and up to 80% for the Myd88 genomic target. Moreover, for the HIV- and MLV-derived nanoblades no cell toxicity and low to undetectable off-target effects were demonstrated.Finally, we also treated hHSCs with nanoblades in combination with an AAV-6 donor encoding vector resulting in over 20% of stable expression cassette knock-in into the WAS gene locus. Currently, we are evaluating these gene modified HSCs for their long-term reconstitution of NOD/SCIDgC-/- mice.Summarizing, this new technology is simple to implement in any laboratory, shows high flexibility for different targets including primary immune cells of murine and human origin, is relatively inexpensive and therefore have important prospects for basic and clinical translation in the area of gene therapy.Fil: Gutierrez, Alejandra. Inserm; FranciaFil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina. Inserm; FranciaFil: Mangeot, Philippe E.. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornellie. Inserm; FranciaFil: Fusil, Floriane. Inserm; FranciaFil: Froment, Gisèle. Inserm; FranciaFil: Martin, Francisco. Inserm; FranciaFil: Bellabdelah, Karim. Universidad de Granada; EspañaFil: Ricci, Emiliano P.. Inserm; FranciaFil: Ayuso, Eduard. Universite de Nantes; FranciaFil: Cosset, François loic. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaAmerican Society of Cell and Gene Therapy 22nd Annual MettingWashingtonEstados UnidosAmerican Society of Cell and Gene Therap

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

Pharmacological induction of a progenitor state for the efficient expansion of primary human hepatocytes

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver

Clearance of Genotype 1b Hepatitis C Virus in Chimpanzees in the Presence of Vaccine-Induced E1-Neutralizing Antibodies

Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection

High TWIST1 mRNA expression is associated with poor prognosis in lymph node-negative and estrogen receptor-positive human breast cancer and is co-expressed with stromal as well as ECM related genes

Introduction: The TWIST homolog 1 (TWIST1) is a transcription factor that induces epithelial to mesenchymal transition (EMT), a key process in metastasis. The purpose of this study was to investigate whether TWIST1 expression predicts disease progression in a large breast cancer cohort with long-term clinical follow-up, and to reveal the biology related to TWIST1 mediated disease progression.Methods: TWIST1 mRNA expression level was analyzed by quantitative real-time reverse polymerase chain reaction (RT-PCR) in 1,427 primary breast cancers. In uni- and multivariate analysis using Cox regression, TWIST1 mRNA expression level was associated with metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS). Separate analyses in lymph node-negative patients (LNN, n = 778) who did not receive adjuvant systemic therapy, before and after stratification into estrogen receptor (ER)-positive (n = 552) and ER-negative (n = 226) disease, were also performed. The association of TWIST1 mRNA with survival endpoints was assessed using Kaplan-Meier analysis. Using gene expression arrays, genes showing a significant Spearman rank correlation with TWIST1 were used to identify overrepresented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-annotated biological pathways.Results: Increased mRNA expression level of TWIST1 analyzed as a continuous variable in both uni- and multivariate analysis was associated with shorter MFS in all patients (hazard ratio (HR): 1.17, 95% confidence interval, (95% CI):1.09 to 1.26; and HR: 1.17, 95% CI: 1.08 to 1.26; respectively), in LNN patients (HR: 1.22, 95% CI: 1.09 to 1.36; and HR: 1.21, 95% CI: 1.07 to 1.36; respectively) and in the ER-positive subgroup of LNN patients (HR: 1.34, 95% CI: 1.17 to 1.53; and HR: 1.32, 95% CI: 1.14 to 1.53; respectively). Similarly, high TWIST1 expression was associated with shorter DFS and OS in all patients and in the LNN/ER-positive subgroup. In contrast, no association of TWIST1 mRNA expression with MFS, DFS or OS was observed in ER-negative patients. Genes h

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

Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease