Biology subjects

Mueller, K.

Publications and source records attributed to Mueller, K..

23 records · Page 2Linked to original sources

bioRxiv2019

Human iPSC modeling elucidates mutation-specific responses to gene therapy in a genotypically diverse dominant maculopathy

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients. SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

genetics↗

bioRxiv2019

Label-free Method for Classification of T cell Activation

T cells have a range of cytotoxic and immune-modulating functions, depending on activation state and subtype. However, current methods to assess T cell function use exogenous labels that often require cell permeabilization, which is limiting for time-course studies of T cell activation and non-destructive quality control of immunotherapies. Label-free optical imaging is an attractive solution. Here, we use autofluorescence imaging of NAD(P)H and FAD, co-enzymes of metabolism, to quantify optical imaging endpoints in quiescent and activated T cells. Machine learning classification models were developed for label-free, non-destructive determination of T cell activation state. T cells were isolated from the peripheral blood of human donors, and a subset were activated with a tetrameric antibody against CD2/CD3/CD28 surface ligands. NAD(P)H and FAD autofluorescence intensity and lifetime of the T cells were imaged using a multiphoton fluorescence lifetime microscope. Significant differences in autofluorescence imaging end-points were observed between quiescent and activated T cells. Feature selection methods revealed that the contribution of the short NAD(P)H lifetime (1) is the most important feature for classification of activation state, across multiple donors and T cell subsets. Logistic regression models achieved 97-99% accuracy for classification of T cell activation from the autofluorescence imaging endpoints. Additionally, autofluorescence imaging revealed NAD(P)H and FAD autofluorescence differences between CD3+CD8+ and CD3+CD4+ T cells, and random forest models of the autofluorescence imaging endpoints achieved 97+% accuracy for four-group classification of quiescent and activated CD3+CD8+ and CD3+CD4+ T cells. Altogether these results indicate that autofluorescence imaging of NAD(P)H and FAD is a powerful method for label-free, non-destructive determination of T cell activation and subtype, which could have important applications for the treatment of cancer, autoimmune, infectious, and other diseases.

bioengineering↗

bioRxiv2018

Medroxyprogesterone acetate mediated alteration in the vaginal microbiota and microenvironment in a Kenyan sex worker cohort

AbstractThe hormonal contraceptive Medroxyprogesterone Acetate (MPA) is associated with increased risk of Human Immunodeficiency Virus (HIV), via incompletely understood mechanisms. Increased diversity in the vaginal microbiota modulates genital inflammation and is associated with increased HIV-1 acquisition. However, the effect of MPA on diversity of the vaginal microbiota is relatively unknown. In a cohort of female Kenyan sex workers, negative for sexually transmitted infections (STIs), with Nugent Scores <7 (N=58 of 370 screened), MPA correlated with significantly increased diversity of the vaginal microbiota as assessed by 16S rRNA gene sequencing. MPA was also significantly associated with low vaginal glycogen and -amylase, factors implicated in vaginal colonization by lactobacilli, bacteria believed to protect against STIs. Furthermore, increased diversity of the vaginal microbiota correlated with activation of vaginal HIV-1 target cells. Results were recapitulated in humanized mice where MPA treatment was associated with increased diversity of the vaginal microbiota, low glycogen, and enhanced HIV-1 susceptibility. Together these results suggest MPA-induced hypo-estrogenism may alter key metabolic components necessary for vaginal colonization by certain bacterial species including lactobacilli, and allow for greater bacterial diversity in the vaginal microbiota. Bacterial diversity in the vaginal microbiota correlates with activation of HIV-1 target cells, which might thus contribute to enhanced susceptibility to HIV-1.

molecular biology↗

bioRxiv2017

Association of Peripheral Blood Pressure with Grey Matter Volume in 19- to 40-Year-Old Adults

ObjectiveTo test whether elevated blood pressure (BP) relates to grey matter volume (GMV) changes in young adults who had not previously been diagnosed as hypertensive (systolic BP (SBP)/diastolic BP (DBP)[≥]140/90 mmHg).\n\nMethodsWe associated BP with GMV from structural 3 Tesla T1-weighted MRI of 423 healthy adults between 19-40 years (mean age=27.7{+/-}5.3 years, 177 women, SBP/DBP=123.2/73.4{+/-}12.2/8.5 mmHg). Data originated from four previously unpublished cross-sectional studies conducted in Leipzig, Germany. We performed voxel-based morphometry on each study separately and combined results in image-based meta-analyses (IBMA) to assess cumulative effects across studies. Resting BP was assigned to one of four categories: (1) SBP<120 and DBP<80 mmHg, (2) SBP 120-129 or DBP 80-84 mmHg, (3) SBP 130-139 or DBP 85-89 mmHg, (4) SBP[≥]140 or DBP[≥]90 mmHg.\n\nResultsIBMA yielded: (a) lower regional GMV was correlated with higher peripheral BP; (b) lower GMV with higher BP when comparing individuals in sub-hypertensive categories 3 and 2, respectively, to those in category 1; (c) lower BP-related GMV was found in regions including hippocampus, amygdala, thalamus, frontal and parietal structures (e.g. precuneus).\n\nConclusionsBP[≥]120/80 mmHg was associated with lower GMV in regions that have previously been related to GM decline in older individuals with manifest hypertension. Our study shows that BP-associated GM alterations emerge continuously across the range of BP and earlier in adulthood than previously assumed. This suggests that treating hypertension or maintaining lower BP in early adulthood might be essential for preventing the pathophysiological cascade of asymptomatic cerebrovascular disease to symptomatic end-organ damage, such as stroke or dementia.

neuroscience↗

bioRxiv2017

Inflated False Negative Rates Undermine Reproducibility In Task-Based fMRI

Reproducibility is generally regarded as a hallmark of scientific validity. It can be undermined by two very different factors, namely inflated false positive rates or inflated false negative rates. Here we investigate the role of the second factor, i.e. the degree to which true effects are not detected reliably. The availability of large public databases and also supercomputing allows us to tackle this problem quantitatively. Specifically, we estimated the reproducibility in task-based fMRI data over different samples randomly drawn from a large cohort of subjects obtained from the Human Connectome Project. We use the full cohort as a standard of reference to approximate true positive effects, and compute the fraction of those effects that was detected reliably using standard software packages at various smaller sample sizes. We found that with standard sample sizes this fraction was less than 25 percent. We conclude that inflated false negative rates are a major factor that undermine reproducibility. We introduce a new statistical inference algorithm based on a novel test statistic and show that it improves reproducibility without inflating false positive rates.

neuroscience↗