Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates

RNAi is a powerful method for suppressing gene expression that has tremendous potential for therapeutic applications. However, because endogenous RNAi plays a role in normal cellular functions, delivery and expression of siRNAs must be balanced with safety. Here we report successful stable expression in primates of siRNAs directed to chemokine (c-c motif) receptor 5 (CCR5) introduced through CD34+ hematopoietic stem/progenitor cell transplant. After hematopoietic reconstitution, to date 14 months after transplant, we observe stably marked lymphocytes expressing siRNAs and consistent down-regulation of chemokine (c-c motif) receptor 5 expression. The marked cells are less susceptible to simian immunodeficiency virus infection ex vivo. These studies provide a successful demonstration that siRNAs can be used together with hematopoietic stem cell transplant to stably modulate gene expression in primates and potentially treat blood diseases such as HIV-1

QUantitative Imaging of eXtraction of oxygen and TIssue consumption (QUIXOTIC) using venular-targeted velocity-selective spin labeling

available in PMC 2012 December 1While oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2) are fundamental parameters of brain health and function, a robust MRI-based mapping of OEF and CMRO2 amenable to functional MRI (fMRI) has not been established. To address this issue, a novel method called QUantitative Imaging of eXtraction of Oxygen and TIssue Consumption, or QUIXOTIC, is introduced. The key innovation in QUIXOTIC is the use of velocity-selective spin labeling to isolate MR signal exclusively from postcapillary venular blood on a voxel-by-voxel basis. Measuring the T2 of this venular-targeted blood allows calibration to venular oxygen saturation (Yv) via theoretical and experimental T2 versus blood oxygen saturation relationships. Yv is converted to OEF, and baseline CMRO2 is subsequently estimated from OEF and additional cerebral blood flow and hematocrit measurements. Theory behind the QUIXOTIC technique is presented, and implications of cutoff velocity (VCUTOFF) and outflow time parameters are discussed. Cortical gray matter values obtained with QUIXOTIC in 10 healthy volunteers are Yv = 0.73 ± 0.02, OEF = 0.26 ± 0.02, and CMRO2 = 125 ± 15 μmol/100 g min. Results are compared to global measures obtained with the T2 relaxation under spin tagging (TRUST) technique. The preliminary data presented suggest that QUIXOTIC will be useful for mapping Yv, OEF, and CMRO2, in both clinical and functional MRI settings.National Institutes of Health (U.S.) (NIH Neuroimaging Training Program Grant, 5-T32-EB001680)National Institutes of Health (U.S.) (NIH Neuroimaging Training Program Grant, 5-R01-EB002066-20)National Institutes of Health (U.S.) (Center for Functional Neuroimaging Technologies; Grant number: P41RR14075S10RR023401)Siemens Aktiengesellschaft (Siemens Medical Solutions)Harvard University--MIT Division of Health Sciences and Technology (Martinos Catalyst Fund)National Cancer Institute (U.S.) (NIH Grant number RO1 EB007942)National Institutes of Health (U.S.) (NIH Medical Scientist Training Program Fellowship, grant no. T32-GM07753

Quantification of normal cerebral oxygen extraction and oxygen metabolism by phase-based MRI susceptometry: evaluation of repeatability using two different imaging protocols.

Global oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2 ) were quantified in a test-retest study. Cerebral blood flow (CBF) data, required for CMRO2 estimation, were obtained using dynamic susceptibility contrast MRI (DSC-MRI). OEF and CMRO2 were quantified using two separate data sets, that is, conventional high-resolution (HR) gradient echo (GRE) phase maps as well as echo planar imaging (EPI) phase maps taken from the baseline (precontrast) part of the DSC-MRI time series. The EPI phase data were included to elucidate whether an extra HR-GRE scan is needed to obtain information about OEF and CMRO2 , or if this information can be extracted from the DSC-MRI experiment only

Identification of genetic alterations in pancreatic cancer by the combined use of tissue microdissection and array-based comparative genomic hybridisation

Pancreatic ductal adenocarcinoma (PDAC) is characterised pathologically by a marked desmoplastic stromal reaction that significantly reduces the sensitivity and specificity of cytogenetic analysis. To identify genetic alterations that reflect the characteristics of the tumour in vivo, we screened a total of 23 microdissected PDAC tissue samples using array-based comparative genomic hybridisation (array CGH) with 1 Mb resolution. Highly stringent statistical analysis enabled us to define the regions of nonrandom genomic changes. We detected a total of 41 contiguous regions (>3.0 Mb) of copy number changes, such as a genetic gain at 7p22.2–p15.1 (26.0 Mb) and losses at 17p13.3–p11.2 (13.6 Mb), 18q21.2–q22.1 (12.0 Mb), 18q22.3–q23 (7.1 Mb) and 18q12.3–q21.2 (6.9 Mb). To validate our array CGH results, fluorescence in situ hybridisation was performed using four probes from those regions, showing that these genetic alterations were observed in 37–68% of a separate sample set of 19 PDAC cases. In particular, deletion of the SEC11L3 gene (18q21.32) was detected at a very high frequency (13 out of 19 cases; 68%) and in situ RNA hybridisation for this gene demonstrated a significant correlation between deletion and expression levels. It was further confirmed by reverse transcription–PCR that SEC11L3 mRNA was downregulated in 16 out of 16 PDAC tissues (100%). In conclusion, the combination of tissue microdissection and array CGH provided a valid data set that represents in vivo genetic changes in PDAC. Our results raise the possibility that the SEC11L3 gene may play a role as a tumour suppressor in this disease

Room temperature chiral magnetic skyrmion in ultrathin magnetic nanostructures

Magnetic skyrmions are chiral spin structures with a whirling configuration. Their topological properties, nanometer size and the fact that they can be moved by small current densities have opened a new paradigm for the manipulation of magnetisation at the nanoscale. To date, chiral skyrmion structures have been experimentally demonstrated only in bulk materials and in epitaxial ultrathin films and under external magnetic field or at low temperature. Here, we report on the observation of stable skyrmions in sputtered ultrathin Pt/Co/MgO nanostructures, at room temperature and zero applied magnetic field. We use high lateral resolution X-ray magnetic circular dichroism microscopy to image their chiral N\'eel internal structure which we explain as due to the large strength of the Dzyaloshinskii-Moriya interaction as revealed by spin wave spectroscopy measurements. Our results are substantiated by micromagnetic simulations and numerical models, which allow the identification of the physical mechanisms governing the size and stability of the skyrmions.Comment: Submitted version. Extended version to appear in Nature Nanotechnolog

Safety of lenadogene nolparvovec gene therapy over 5 years in 189 patients with Leber hereditary optic neuropathy

Purpose: Evaluate the safety profile of lenadogene nolparvovec (Lumevoq®) in patients with Leber hereditary optic neuropathy. Design: Pooled analysis of safety data from 5 clinical studies. Methods: A total of 189 patients received single unilateral or bilateral intravitreal injections of a recombinant adeno-associated virus 2 (rAAV2/2) vector encoding the human wild-type ND4 gene. Adverse events (AEs) were collected throughout the studies, up to 5 years. Intraocular inflammation and increased intraocular pressure (IOP) were ocular AEs of special interest. Other assessments included ocular examinations, vector bio-dissemination and systemic immune responses against rAAV2/2. Results: Almost all patients (95.2%) received 9 × 1010 viral genomes and 87.8% had at least 2 years of follow-up. Most patients (75.1%) experienced at least one systemic AE, but systemic treatment-related AEs occurred in 3 patients, none was serious. Intraocular inflammation was reported in 75.6% of lenadogene nolparvovec-treated eyes. Almost all intraocular inflammations occurred in the anterior chamber (58.8%) or in the vitreous (40.3%) and was of mild (90.3%) or moderate (8.8%) intensity; most resolved with topical corticosteroids alone. All IOP increases were mild to moderate in intensity. No AE led to study discontinuation. Bio-dissemination of lenadogene nolparvovec and systemic immune response were limited. The safety profile was comparable for patients treated bilaterally and unilaterally. Conclusions: Lenadogene nolparvovec has a good overall safety profile with excellent systemic tolerability, consistent with limited bio-dissemination. The product is well tolerated, with mostly mild ocular side effects responsive to conventional ophthalmologic treatments

Distinct Genomic Integration of MLV and SIV Vectors in Primate Hematopoietic Stem and Progenitor Cells

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+) cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors

Solanum lycopersicon Mill. and Nicotiana benthamiana L. under high light show distinct responses to anti-oxidative stress

Two experimentally important species, Solanum lycopersicon Mill. and Nicotiana benthamiana L., were propagated in vitro under low light (50 mmolm 2 s 1) and transferred to HL (200 mmolm 2 s 1) under a protocol previously developed for grapevine and chestnut. Compared with photooxidative stress parameters already tested in those species, imaging of hydrogen peroxide and superoxide revealed an accumulation on d2–3 and d6 in S. lycopersicon and d1–2 and d5–7 in N. benthamiana. SOD, CAT and APX activities matched ROS accumulation. The expression of the respective transcripts showed a significant increase on d1 in S. lycopersicon while in N. benthamiana a bimodal pattern was found, with peaks on d2 and d7. These results, together with the relative timing of root expansion and new leaf emergence, indicate that these two apparently similar species display different strategies when responding to light stress, evidencing further the uniqueness of the response of each species. The behaviour of N. benthamiana falls closely into the pattern already reported for wood species including grapevin