Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B

BACKGROUND: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX). METHODS: Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8(+) T cell responses to hF.IX. RESULTS: Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8(+) T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome. CONCLUSIONS: The nature of the AAV genome can impact the CD8(+) T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome

Innate Immune Responses to AAV Vectors

Gene replacement therapy by in vivo delivery of adeno-associated virus (AAV) is attractive as a potential treatment for a variety of genetic disorders. However, while AAV has been used successfully in many models, other experiments in clinical trials and in animal models have been hampered by undesired responses from the immune system. Recent studies of AAV immunology have focused on the elimination of transgene-expressing cells by the adaptive immune system, yet the innate immune system also has a critical role, both in the initial response to the vector and in prompting a deleterious adaptive immune response. Responses to AAV vectors are primarily mediated by the TLR9–MyD88 pathway, which induces the production of pro-inflammatory cytokines by activating the NF-κB pathways and inducing type I IFN production; self-complementary AAV vectors enhance these inflammatory processes. Additionally, the alternative NF-κB pathway influences transgene expression in cells transduced by AAV. This review highlights these recent discoveries regarding innate immune responses to AAV and discusses strategies to ablate these potentially detrimental signaling pathways

Low-frequency ventilation during cardiopulmonary bypass for lung protection:A randomized controlled trial

OBJECTIVE: Pulmonary dysfunction is a common complication in patients undergoing heart surgery. Current clinical practice does not include any specific strategy for lung protection. To compare the anti-inflammatory effects of low-frequency ventilation (LFV), as measured by nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) p65 pathway activation, for the entire cardiopulmonary bypass (CPB) vs both lungs left collapsed in patients undergoing coronary artery bypass grafting (CABG). METHODS: Two groups parallel randomized controlled trial. The primary outcome was inflammation measured by NF-κB p65 activation in pre- and post-CPB lung biopsies. Secondary outcomes were additional inflammatory markers in both biopsy tissue and blood. RESULTS: Thirty-seven patients were randomly allocated to LFV (18) and to both lungs left collapsed (19). The mean concentration of NF-κB p65 in the biopsies before chest closure (adjusted for pre-CPB concentration) was higher in the LFV group compared to both lungs left collapsed group but this was not significant (0.102, 95% confidence interval, -0.022 to 0.226, P = 0.104). There were no significant differences between groups in the other inflammatory markers measured in tissue and blood. CONCLUSIONS: In patients undergoing elective CABG, the use of LFV during CPB when compared to both lungs left collapsed does not seem to reduce inflammation in lung biopsies and blood

230 GHz VLBI OBSERVATIONS OF M87: EVENT‐HORIZON‐SCALE STRUCTURE DURING AN ENHANCED VERY‐HIGH‐ENERGY γ‐RAY STATE IN 2012

We report on 230 GHz (1.3 mm) very long baseline interferometry (VLBI) observations of M87 with the Event Horizon Telescope using antennas on Mauna Kea in Hawaii, Mt. Graham in Arizona, and Cedar Flat in California. For the first time, we have acquired 230 GHz VLBI interferometric phase information on M87 through measurement of the closure phase on the triangle of long baselines. Most of the measured closure phases are consistent with 0° as expected by physically motivated models for 230 GHz structure such as jet models and accretion disk models. The brightness temperature of the event-horizon-scale structure is ~1 X 10[superscript 10] K derived from the compact flux density of ~1 Jy and the angular size of ~40 µas ~ 5.5 R[subscript s], which is broadly consistent with the peak brightness of the radio cores at 1–86 GHz located within ~10[superscript 2] R[subscript s]. Our observations occurred in the middle of an enhancement in very-high-energy (VHE) γ-ray flux, presumably originating in the vicinity of the central black hole. Our measurements, combined with results of multi-wavelength observations, favor a scenario in which the VHE region has an extended size of ~20–60 R[subscript s]

1.3 mm Wavelength VLBI of Sagittarius A*: Detection of Time-Variable Emission on Event Horizon Scales

Sagittarius A*, the ~4 x 10^6 solar mass black hole candidate at the Galactic Center, can be studied on Schwarzschild radius scales with (sub)millimeter wavelength Very Long Baseline Interferometry (VLBI). We report on 1.3 mm wavelength observations of Sgr A* using a VLBI array consisting of the JCMT on Mauna Kea, the ARO/SMT on Mt. Graham in Arizona, and two telescopes of the CARMA array at Cedar Flat in California. Both Sgr A* and the quasar calibrator 1924-292 were observed over three consecutive nights, and both sources were clearly detected on all baselines. For the first time, we are able to extract 1.3 mm VLBI interferometer phase information on Sgr A* through measurement of closure phase on the triangle of baselines. On the third night of observing, the correlated flux density of Sgr A* on all VLBI baselines increased relative to the first two nights, providing strong evidence for time-variable change on scales of a few Schwarzschild radii. These results suggest that future VLBI observations with greater sensitivity and additional baselines will play a valuable role in determining the structure of emission near the event horizon of Sgr A*.Comment: 8 pages, submitted to ApJ

Computed tomographic evaluation of the distal limb in the standing sedated horse : technique, imaging diagnoses, feasibility, and artifacts

In several veterinary institutions, adjustments of CT machines have been made that allow for imaging of the standing horse. The risk of general anesthesia is eliminated and the shorter scan completion time reduces cost to clients. The objective of this retrospective, analytical study was to evaluate the technique, imaging diagnoses, feasibility, and image artifacts of multi-slice helical CT of horses’ distal limbs acquired under standing sedation. The CT images of 250 horses of various breeds, aged 3–23 years, that underwent standing distal limb CT were evaluated. Three observers assessed the CT images for artifacts and inter-observer agreement was calculated. Eighty-six percent (95% confidence interval (CI), 81–90) of the scans were carried out on the forelimbs, while 14% (95% CI, 10–19) were of the hindlimbs. A total of 65% (95% CI, 59–71) of horses that underwent standing sedated CT had single imaging diagnoses. Seventy-one percent (95% CI, 65–77) of the cases had unilateral lesions, 27% (95% CI, 22–33) had bilateral lesions and 2% (95% CI, 1–4) had no diagnosed lesions. The average CT acquisition time was 17.5 minutes (range = 15–20). The average number of acquisitions per horse was 1.7 (median = 1; range = 1–4). There was good to excellent agreement between all three observers for the presence of motion artifact in the metacarpo/metatarsophalangeal joints, identification of marked beam hardening artifact, mild solar/ skin dirt, and photon starvation artifact (kappa 0.61-0.80). No complications were encountered. Standing examination of the distal limb achieved diagnostic image quality that was obtained with minimal acquisition attempts and in a timely manner.DATA AVAILABILITY STATEMENT: Data available at https://doi.org/10.25403/UPresearchdata.18550880.v1.https://wileyonlinelibrary.com/journal/vruhj2022Companion Animal Clinical StudiesProduction Animal Studie

Detection of intrinsic source structure at ~3 Schwarzschild radii with Millimeter-VLBI observations of SAGITTARIUS A*

We report results from very long baseline interferometric (VLBI) observations of the supermassive black hole in the Galactic center, Sgr A*, at 1.3 mm (230 GHz). The observations were performed in 2013 March using six VLBI stations in Hawaii, California, Arizona, and Chile. Compared to earlier observations, the addition of the APEX telescope in Chile almost doubles the longest baseline length in the array, provides additional {\it uv} coverage in the N-S direction, and leads to a spatial resolution of $\sim$30 $\mu$as ($\sim$3 Schwarzschild radii) for Sgr A*. The source is detected even at the longest baselines with visibility amplitudes of $\sim$4-13% of the total flux density. We argue that such flux densities cannot result from interstellar refractive scattering alone, but indicate the presence of compact intrinsic source structure on scales of $\sim$3 Schwarzschild radii. The measured nonzero closure phases rule out point-symmetric emission. We discuss our results in the context of simple geometric models that capture the basic characteristics and brightness distributions of disk- and jet-dominated models and show that both can reproduce the observed data. Common to these models are the brightness asymmetry, the orientation, and characteristic sizes, which are comparable to the expected size of the black hole shadow. Future 1.3 mm VLBI observations with an expanded array and better sensitivity will allow a more detailed imaging of the horizon-scale structure and bear the potential for a deep insight into the physical processes at the black hole boundary.Comment: 11 pages, 5 figures, accepted to Ap