Biology subjects

Lee, B.-C.

Publications and source records attributed to Lee, B.-C..

7 recordsLinked to original sources

bioRxiv2023

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34+ cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34+ cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP+ cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

cell biology↗

bioRxiv2023

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection. HighlightsO_LINo evidence of association between CH and COVID-19 clinical severity in macaques. C_LIO_LIThe presence of CH is associated with prolonged local inflammatory responses in COVID-19. C_LIO_LISARS-CoV-2 persists longer in respiratory tracts of macaques with CH following infection. C_LI

genetics↗

bioRxiv2021

Strategic infarct locations for post-stroke depressive symptoms: a lesion- and disconnection-symptom mapping study

BackgroundDepression is the most common neuropsychiatric complication after stroke. Infarct location is associated with post-stroke depressive symptoms (PSDS), but it remains debated which brain structures are critically involved. We performed a large-scale lesion-symptom mapping study to identify infarct locations, and white matter disconnections, associated with PSDS. MethodsWe included 553 patients (age 69{+/-}11 years, 42% female) with acute ischemic stroke. PSDS were measured using the 30-item Geriatric Depression Scale (GDS-30). Multivariable support vector regression (SVR)-based analyses were performed both at the level of individual voxels (SVR-VLSM) and predefined regions of interest (SVR-ROI) to relate infarct location to PSDS. We externally validated our findings in an independent stroke cohort (N=459). Finally, disconnectome-based analyses were performed using SVR-VLSM, in which white matter fibers disconnected by the infarct were analyzed instead of the infarct itself. Results: Infarcts in the right amygdala, right hippocampus and right pallidum were consistently associated with PSDS (permutation-based p<0.05) in SVR-VLSM and SVR-ROI. External validation (N=459) confirmed the association between infarcts in the right amygdala and pallidum, but not the right hippocampus, and PSDS. Disconnectome-based analyses revealed that disconnections in the right parahippocampal white matter, right thalamus and pallidum, and right anterior thalamic radiation were significantly associated (permutation-based p<0.05) with PSDS. ConclusionsInfarcts in the right amygdala and pallidum, and disconnections of right limbic and frontal cortico-basal ganglia-thalamic circuits, are associated with PSDS. Our findings provide a comprehensive and integrative picture of strategic infarct locations for PSDS, and shed new light on pathophysiological mechanisms of depression after stroke.

neuroscience↗

bioRxiv2020

A novel PS4 criterion approach based on symptoms of rare diseases and in-house frequency data in a Bayesian framework.

The American College of Medical Genetics (ACMG) and Genomics/Association for Molecular Pathology (AMP) previously reported standardized guidance for the assessment of genetic variants. One of the criteria regarding the prevalence in a case-control study, PS4, is important due to its evidence of pathogenicity. Despite recent studies approaching gene- and disease-specific probands, interpretation of a variant to PS4 still has certain limitations for rare variants. Here, we suggest a generalized method, Bayesian odds ratio (BayesianOR), applicable to PS4 via decomposing a disease to its symptoms and applying a Bayesian framework. Using this approach, we demonstrate reproducibility of the calculation of the original odds ratio from well-studied epilepsy data and verify the applicability to in-house frequencies for various rare diseases. In addition, BayesianOR showed a significant difference in tendency with different ClinVar pathogenicity, using in-house data. Thus, the novel method described here should provide an improved interpretation of sequence variants. Furthermore, we anticipate that it will enhance the diagnosis of patients with rare diseases.

genetics↗

bioRxiv2020

Validation and Long-Term Follow Up of CD33 Off-Targets Predicted In Vitro and In Silico Using Error-Corrected Sequencing in Rhesus Macaques

ABSTRACTThe programmable nuclease technology CRISPR/Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR/Cas9 therapeutically. Here, we utilized a rhesus macaque model to ask whether CIRCLE-Seq (CS), an in vitro off-target prediction method, more accurately identifies off-targets compared to in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-Seq and ISP for guide RNAs designed against TET2 and CD33 genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both ISP and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a CD33 gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both ISP and CIRCLE-Seq, however there was poor correlation between the sites predicted by the two methods. When almost 500 sites identified by each method were searched for in hematopoietic cells following transplantation, 19 detectable mutations in off-target sites were detected via error-corrected sequencing. Of these 19 sites, 8 sites were predicted in the top 500 sites by both methods, 8 by CIRCLE-Seq only, and 3 by ISP only. Cells with off-target editing exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In conclusion, neither methodology predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods may be required for optimizing safety of clinical development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Kinematics associated with treadmill walking in Rett Syndrome

Individuals with Rett syndrome suffer from severely impaired cognitive and motor performance. Current movement-related therapeutic programs often include traditional physical therapy activities and assisted treadmill walking routines for those patients who are ambulatory. However, there are no quantitative reports of kinematic gait parameters obtained during treadmill walking. Here we report the results of an investigation of 17 females diagnosed with typical Rett who walked on a treadmill as speed gradually increased. The objective included characterizing lower limb kinematics, including knee and hip joint range of motions, velocities, limb asymmetries, and the variance associated with these measures. Joint kinematics were obtained using a 12 camera motion capture system and associated processing and analysis software. Stride times progressively decreased as treadmill speeds increased although the range of speeds our participant could walk was quite slow: range 0.2 m/s - 0.5 m/s. There were significant main effects of speed on sagittal knee and hip range of motions and hip velocity. There were large joint asymmetries and variance values relative to both healthy walkers and others patient populations although variance values decreased as walking speed increased. There were significant correlations between joint range of motions and stride times and joint velocities and stride times. The results indicate that Rett patients can adapt their kinematic gait patterns in response to increasing treadmill speed but their ability to do so lies within a narrow range of speeds. We suggest that treadmill training for ambulatory individuals with Rett may further promote improved walking kinematics as well as overall health benefits.

neuroscience↗

bioRxiv2018

Cryo-EM structures reveal bilayer remodeling during Ca2+ activation of a TMEM16 scramblase

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several members of the TMEM16 family have been identified as Ca2+-activated scramblases but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs, revealing large Ca2+-dependent conformational changes of the protein as well as significant, function dependent membrane reorganization.

biochemistry↗