AAV2-Mediated Combined Subretinal Delivery of IFN-α and IL-4 Reduces the Severity of Experimental Autoimmune Uveoretinitis

We previously showed that adeno-associated virus 2 (AAV2) mediated subretinal delivery of human interferon-alpha (IFN-α) could effectively inhibit experimental autoimmune uveoretinitis (EAU). In this study we investigated whether subretinal injection of both AVV2.IFN-α and AAV2.IL-4 had a stronger inhibition on EAU activity. B10RIII mice were subretinally injected with AAV2.IFN-α alone (1.5×107 vg), AAV2.IL-4 alone (3.55×107 vg), and AAV2.IFN-α combined with AAV2.IL-4. PBS, AAV2 vector encoding green fluorescent protein (AAV2.GFP) (5×107 vg) was subretinally injected as a control. IFN-α and IL-4 were effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2 vectors either alone or following combined administration and significantly attenuated EAU activity clinically and histopathologically. AAV2.IL-4 showed a better therapeutic effect as compared to AAV2.IFN-α. The combination of AAV2.IL-4 and AAV2.IFN-α was not significantly different as compared to AAV2.IL-4 alone. There was no difference concerning DTH (delayed-type hypersensitivity) reaction, lymphocyte proliferation and IL-17 production among the investigated treatment groups, suggesting that local retinal gene delivery did not affect the systemic immune response

LSD1 inhibition attenuates androgen receptor V7 splice variant activation in castration resistant prostate cancer models

Background: Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7. Methods: We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7. Results: Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments. Conclusion: LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted

55 Cancri e's occultation captured with CHEOPS

Past occultation and phase-curve observations of the ultra-short period super-Earth 55 Cnc e obtained at visible and infrared wavelengths have been challenging to reconcile with a planetary reflection and emission model. In this study, we analyse a set of 41 occultations obtained over a two-year timespan with the CHEOPS satellite. We report the detection of 55 Cnc e's occultation with an average depth of $12\pm3$ ppm. We derive a corresponding 2-$\sigma$ upper limit on the geometric albedo of $A_g < 0.55$ once decontaminated from the thermal emission measured by Spitzer at 4.5$\mu$m. CHEOPS's photometric performance enables, for the first time, the detection of individual occultations of this super-Earth in the visible and identifies short-timescale photometric corrugations likely induced by stellar granulation. We also find a clear 47.3-day sinusoidal pattern in the time-dependent occultation depths that we are unable to relate to stellar noise, nor instrumental systematics, but whose planetary origin could be tested with upcoming JWST occultation observations of this iconic super-Earth.Comment: In press. Accepted for publication in Astronomy and Astrophysics on 13 October 2022. 10 pages, 7 figures and 3 table

Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure–function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. “Shielding” polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating ‘designer’ gene delivery vectors with enhanced properties

55 Cancri e's occultation captured with CHEOPS

peer reviewe

Analytical optimization and test validation of the submicron dimensional stability of the CHEOPS space telescope's CFRP structure

The CHEOPS (CHaracterising ExOPlanet Satellite), which is an ESA mission developed in cooperation with Switzerland and a number of other member-states, is the first one dedicated to search for transits by means of ultrahigh precision photometry on bright stars already known to host planets. The optical design is based on a Ritchey-Chretien style telescope to provide a de-focussed image of the target stars. The telescope’s mirrors M1, M2 as well as the focal plane detector are supported by a thermally controlled CFRP structure suspended on isostatic mounts. The dimensional stability of the structural system supporting the optics is a key requirement as it directly impacts the instrument’s accuracy. The M1 and M2 mirrors are supported by a tubular CFRP telescope design which has been optimized by analyses down to carbon fibre layer level with the support of extensive sample test results for model correlation and accurate dimensional stability predictions. This sample characterization test campaign has been conducted on samples with different carbon fibre layups (orientation and stack sequence) to measure accurately the Coefficient of Thermal Expansion (CTE) over a wide temperature range extending from -80°C to +80°C. Using the correlated Finite Element Model, the fibre orientation layup that minimized the relative displacement between the M1 and M2 mirrors, including the consideration of the thermo-elastic contributions of the isostatic mounts on the overall stability of this optical system, has been identified and selected for the baseline design of the CHEOPS Structure. A dedicated Structural and Thermal Model (STM2), which was then refurbished to a PFM, was manufactured and tested with an ad hoc setup to verify the overall structural stability of the optical train assembly [2]. The relative distance between M1 and M2 was measured under thermal vacuum conditions using laser interferometer techniques. Thermal cycling tests were initially conducted to eliminate and characterize settling effects. Then, the structure’s stability was measured at three stabilised operational temperatures: -5, -10 and -15°C. The thermally induced M1-M2 misalignment on the optical axis was measured to be between -0.156 and -0.168 micron/°C. Relative mirror tilt and lateral centre shifts were also measured. The obtained focal distance, tilt and centre shift stability between mirrors M1 and M2 were all compliant with the system level requirements such that both an STM and PFM model of the CHEOPS CFRP Structure were successfully qualified and delivered in due time for integration on the spacecraft

Methods for the in vitro determination of an individual disposition towards TH1- or TH2-reactivity by the application of appropriate stimulatory antigens

In this study we performed several methods for the determination of cytokines (RT-PCR for the demonstration of cytokine mRNA and flow cytometry for the analysis of intracellular cytokines) and compared them with a recently established test system stimulating peripheral blood mononuclear cells (PBMC) with TH1- and TH2-relevant recall antigens and analysing type 1 and type 2 cytokines by ELISA. Aim of the study was therefore to evaluate the reliability of TH1/TH2 cytokine profiles in two individuals with different types of an allergic/atopic disposition: one of them showed a strong TH1/type 1-mediated tuberculin-reaction (subject A), the other (subject B) revealed elevated IgE-levels and eosinophil counts (TH2/type 2-mediated). PBMC were incubated with the type 1-antigen purified protein derivative (PPD) and the type 2-antigen tetanus-toxoid (TT) for seven days. From the comparison of ELISA with RT-PCR and flow cytometry-analysis it became evident that all three methods allowed the definition of subject A as a ‘type 1-responder’. Subject B showed a pure type 2-response in the ELISA method; PCR and flow cytometry analysis revealed the simultaneous production of type 1- and type 2-cytokines resulting in a mixed type 1/type 2-profile. Active immunization of subject A with TT at the end of the observation period of 12 months resulted in a transient shift from type 1- to a mixed type 1/type 2-profile (simultaneous PPD-induced IFN-γ- and TT-induced IL-5 production). From this pilot study based on clear cut clinical criteria concerning either a humoral or cellular immunological reactivity towards allergens/antigens it is suggested that the determination of type 1/type 2-cytokines by ELISA in supernatants of PBMC stimulated with type 1/type 2-relevant antigens is a useful approach for a better classification of ‘type1-’ or ‘type 2-responder’

Prediction of adeno-associated virus neutralizing antibody activity for clinical application

Patients with neutralizing antibodies (Nab) against adeno-associated virus (AAV) are usually excluded from treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including: sera volume, AAV dose/cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a 4-fold increase in sensitivity for measurement of Nab titers was observed compared to an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various Phase I clinical trials