Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy.

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal. Here, we present and showcase a machine learning (ML) method for designing AAV peptide insertion libraries that achieve fivefold higher packaging fitness than the standard NNK library with negligible reduction in diversity. To demonstrate our ML-designed library's utility for downstream engineering goals, we show that it yields approximately 10-fold more successful variants than the NNK library after selection for infection of human brain tissue, leading to a promising glial-specific variant. Moreover, our design approach can be applied to other types of libraries for AAV and beyond.

Anxiety-related behavior and associated brain transcriptome and epigenome alterations in adult female zebrafish exposed to atrazine during embryogenesis.

Atrazine often contaminates drinking water sources, exceeding the maximum contaminant level established by the US Environmental Protection Agency at 3 parts per billion (ppb; µg/L). Atrazine is linked to endocrine disruption, neurotoxicity, and cancer, with delayed health effects observed after developmental exposure in line with the developmental origins of health and disease (DOHaD) hypothesis. To test the hypothesis that embryonic atrazine exposure induces delayed neurotoxicity in adult female zebrafish (Danio rerio), embryos were exposed to 0, 0.3, 3, or 30 ppb atrazine during embryogenesis (1-72 h post fertilization (hpf)) and raised to adults with no additional atrazine exposure. Behavioral outcomes were tested through a novel tank test, light-dark box, and open field test and indicated female zebrafish had more anxious phenotypes at 9 months post fertilization (mpf). Female brain transcriptomic analysis at 9 mpf found altered gene expression pathways related to organismal injury and cancer with beta-estradiol and estrogen receptor as top upstream regulators. These results were compared to 9 mpf male and 6 mpf female groups with the same atrazine embryonic exposures and showed differences in specific genes that were altered, but similarities in top molecular pathways. Molecular pathways associated with behavior were observed only in the 6 mpf transcriptomic profiles, suggesting prediction of observed behavioral outcomes at 9 mpf. The expression of genes associated with serotonin neurotransmission was also evaluated at 14 mpf to determine persistence; however, no significant changes were observed. Brain global methylation in 12 mpf zebrafish observed an increased percent 5 mC in females with embryonic 0.3 ppb atrazine exposure. Finally, the body length, body weight, and brain weight were determined at 14 mpf and were altered in all treatment groups. These results indicate that embryonic atrazine exposure does cause delayed neurotoxicity within the DOHaD framework, which is significant given atrazine's presence and persistence in the environment.

Atrazine exposure in zebrafish induces aberrant genome-wide methylation.

Atrazine (ATZ) is the second most common agricultural herbicide used in the United States and is an endocrine disrupting chemical (EDC). Developmental exposure to ATZ can lead to significant behavioral and morphological alterations in exposed animals and their progeny suggesting the involvement of an epigenetic mechanism. Specific epigenetic mechanisms responsible for these alterations, however, are yet to be elucidated. In this study, we exposed zebrafish embryos to 0, 0.3, 3, or 30 ppb (µg/L) of ATZ from 1 to 72 h post fertilization (hpf). Chemical exposure was ceased and zebrafish maintained until 9 months post fertilization (mpf), when whole-genome bisulfite sequencing (WGBS) was performed to assess the effects of embryonic ATZ exposure on DNA methylation in female fish brains. The number of differentially methylated genes (DMGs) increased with increasing treatment concentration. DMGs were enriched in neurological pathways with extensive methylation changes consistently observed in neuroendocrine pathways. Specifically, DMGs with methylation changes in promoter regions showed hypomethylation in estrogen receptor signaling and hypermethylation in androgen signaling. DMGs with methylation changes in genebody were primarily enriched for mitochondrion-related pathways associated with healthy aging. Integrated analysis with transcriptomic data at 9 mpf exhibited a similar trend identifying CABLES1 and NDUFA4 as shared targets at all concentrations. We then compared the predicted upstream regulators of transcriptomic changes with DMGs and identified CALML3 as a common upstream regulator at both 0.3 and 30 ppb that exhibit significant methylation changes. Collectively, our study identified long-lasting DNA methylation changes in genome after embryonic ATZ exposure and elucidated potential gene targets whose aberrant methylation features may drive alterations in gene transcription in long-term.

Single-Cell Image-Based Analysis Reveals Chromatin Changes during the Acquisition of Tamoxifen Drug Resistance.

Cancer drug resistance is the leading cause of cancer related deaths. The development of drug resistance can be partially contributed to tumor heterogeneity and epigenetic plasticity. However, the detailed molecular mechanism underlying epigenetic modulated drug resistance remains elusive. In this work, we systematically analyzed epigenetic changes in tamoxifen (Tam) responsive and resistant breast cancer cell line MCF7, and adopted a data-driven approach to identify key epigenetic features distinguishing between these two cell types. Significantly, we revealed that DNA methylation and H3K9me3 marks that constitute the heterochromatin are distinctively different between Tam-resistant and -responsive cells. We then performed time-lapse imaging of 5mC and H3K9me3 features using engineered probes. After Tam treatment, we observed a slow transition of MCF7 cells from a drug-responsive to -resistant population based on DNA methylation features. A similar trend was not observed using H3K9me3 probes. Collectively, our results suggest that DNA methylation changes partake in the establishment of Tam-resistant breast cancer cell lines. Instead of global changes in the DNA methylation level, the distribution of DNA methylation features inside the nucleus can be one of the drivers that facilitates the establishment of a drug resistant phenotype in MCF7.

Lead exposure induces dysregulation of constitutive heterochromatin hallmarks in live cells.

Lead (Pb) is a heavy metal contaminant commonly found in air, soil, and drinking water due to legacy uses. Excretion of ingested Pb can result in extensive kidney damages due to elevated oxidative stress. Epigenetic alterations induced by exposure to Pb have also been implied but remain poorly understood. In this work, we assessed changes in repressive epigenetic marks, namely DNA methylation (meCpG) and histone 3 lysine 9 tri-methylation (H3K9me3) after exposure to Pb. Live cell epigenetic probes coupled to bimolecular fluorescence complementation (BiFC) were used to monitor changes in the selected epigenetic marks. Exposure to Pb significantly lowered meCpG and H3K9me3 levels in HEK293T cells suggesting global changes in constitutive heterochromatin. A heterodimeric pair of probes that tags chromatin regions enriched in both meCpG and H3K9me3 further confirmed our findings. The observed epigenetic changes can be partially attributed to aberrant transcriptional changes induced by Pb, such as overexpression of TET1 after Pb exposure. Lastly, we monitored changes in selected heterochromatin marks after removal of Pb and found that changes in these markers do not immediately recover to their original level suggesting potential long-term damages to chromatin structure.

Low dose lead exposure induces alterations on heterochromatin hallmarks persisting through SH-SY5Y cell differentiation.

Lead (Pb) is a commonly found heavy metal due to its historical applications. Recent studies have associated early-life Pb exposure with the onset of various neurodegenerative disease. The molecular mechanisms of Pb conferring long-term neurotoxicity, however, is yet to be elucidated. In this study, we explored the persistency of alteration in epigenetic marks that arise from exposure to low dose of Pb using a combination of image-based and gene expression analysis. Using SH-SY5Y as a model cell line, we observed significant alterations in global 5-methycytosine (5 mC) and histone 3 lysine 27 tri-methylation (H3K27me3) and histone 3 lysine 9 tri-methylation (H3K9me3) levels in a dose-dependent manner immediately after Pb exposure. The changes are partially associated with alterations in epigenetic enzyme expression levels. Long term culturing (14 days) after cease of exposure revealed persistent changes in 5 mC, partial recovery in H3K9me3 and overcompensation in H3K27me3 levels. The observed alterations in H3K9me3 and H3K27me3 are reversed after neuronal differentiation, while reduction in 5 mC levels are amplified with significant changes in patterns as identified via texture clustering analysis. Moreover, correlation analysis demonstrates a strong positive correlation between trends of 5 mC alteration after differentiation and neuronal morphology. Collectively, our results suggest that exposure to low dose of Pb prior to differentiation can result in persistent epigenome alterations that can potentially be responsible for the observed phenotypic changes. Our work reveals that Pb induced changes in epigenetic repressive marks can persist through neuron differentiation, which provides a plausible mechanism underlying long-term neurotoxicity associated with developmental Pb-exposure.

Embryonic atrazine exposure and later in life behavioral and brain transcriptomic, epigenetic, and pathological alterations in adult male zebrafish.

Atrazine (ATZ), a commonly used pesticide linked to endocrine disruption, cancer, and altered neurochemistry, frequently contaminates water sources at levels above the US Environmental Protection Agency's 3 parts per billion (ppb; µg/L) maximum contaminant level. Adult male zebrafish behavior, brain transcriptome, brain methylation status, and neuropathology were examined to test the hypothesis that embryonic ATZ exposure causes delayed neurotoxicity, according to the developmental origins of health and disease paradigm. Zebrafish (Danio rerio) embryos were exposed to 0 ppb, 0.3 ppb, 3 ppb, or 30 ppb ATZ during embryogenesis (1-72 h post fertilization (hpf)), then rinsed and raised to maturity. At 9 months post fertilization (mpf), males had decreased locomotor parameters during a battery of behavioral tests. Transcriptomic analysis identified altered gene expression in organismal development, cancer, and nervous and reproductive system development and function pathways and networks. The brain was evaluated histopathologically for morphometric differences, and decreased numbers of cells were identified in raphe populations. Global methylation levels were evaluated at 12 mpf, and the body length, body weight, and brain weight were measured at 14 mpf to evaluate effects of ATZ on mature brain size. No significant difference in genome methylation or brain size was observed. The results demonstrate that developmental exposure to ATZ does affect neurodevelopment and neural function in adult male zebrafish and raises concern for possible health effects in humans due to ATZ's environmental presence and persistence. Graphical abstract.

Feasibility of using the International Classification of Functioning, Disability and Health Core Set for evaluation of fall-related risk factors in acute rehabilitation settings.

BACKGROUND: Previously, we reported the use of an International Classification of Functioning (ICF) core set that can provide a holistic framework for evaluating the risk factors of falls; however, data on the feasibility of applying this core set are lacking. AIM: To investigate the feasibility of applying the fall-related ICF risk-factor core set in the case of patients in an acute-rehabilitation setting. DESIGN: A cross-sectional and descriptive correlational design. SETTING: Acute-rehabilitation ward. POPULATION: A total of 273 patients who experienced fall at acute-rehabilitation ward. METHODS: The data on falls were collected from the hospital's Nursing Information System (NIS) and the fall-reporting system (Adverse Event Reporting System, AERS) between 2010 and 2013. The relationship of both systems to the fall-related ICF core set was analyzed to assess the feasibility of their clinical application. We evaluated the feasibility of using the fall-related ICF risk-factor core set by using the frequency and the percentage of the fall patients in of the listed categories. RESULTS: The fall-related ICF risk-factor core set category b735 (muscle tone functions) exhibited a high feasibility (85.95%) for clinical application, and the category b730 (muscle power functions) covered 77.11% of the patients. The feasibility of application of the category d410 (change basic body position) was also high in the case of all fall patients (81.69%). CONCLUSIONS: In the acute-rehabilitation setting, the feasibility of application of the fall-related ICF risk-factor core set is high. CLINICAL REHABILITATION IMPACT: The fall-related ICF risk-factor core set can help multidisciplinary teams develop fall-prevention strategies in acute rehabilitation wards.

Dopamine transporter density by [99mTc]-TRODAT-1 SPECT and neurocognitive performance - a preliminary pilot study

Introducción: los déficits cognitivos están relacionados con el deterioro funcional y con la baja calidad de vida en la enfermedad de Parkinson (EP). El sistema dopaminérgico de los ganglios basales es importante para el funcionamiento cognitivo y motor. Radiomarcadores de transportador de Dopamina (TAD) han sido utilizados para calcular la pérdida neuronal dopaminérgica en humanos. Objetivos: estudiar la relación entre el deterioro cognitivo y la pérdida neuronal dopaminérgica estriatal en pacientes con EP. Métodos: quince pacientes fueron escaneados con [99mTc]-TRODAT-1 y SPECT. El estriado (STR) y el lóbulo occipital (BKG) fueron definidos como regiones de interés (RIs) para la obtención del potencial de ligación (PL = [STR - BKG] / BKG). Exámenes neurocognitivos fueron aplicados, incluyendo el Rey Auditory Verbal Learning Test (RAVLT), Wisconsin Card Sorting Test (WCST), Ravens Progressive Matrices, Digit Span y Tavis 3. Resultados: El PL fue correlacionado negativamente con los exámenes de RAVLT 4 y 5, que evalúan el aprendizaje verbal. El PL también fue correlacionado negativamente con el artículo de aprendizaje de WCST y los artículos de Tavis 3, el error de acción y el número de aciertos. Conclusiones: este estudio indica que la pérdida de TAD estriatal está asociada con un desempeño mas pobre en tareas de flexibilidad cognitiva y aprendizaje verbal. Estos resultados están de acuerdo con un estudio previo con participantes sanos que encontró una relación entre la densidad de TAD del caudado y el desempeño en tareas de aprendizaje verbal. La segmentación del caudado/putamen en una muestra mayor está en desarrollo y podrá proveer más información sobre déficits cognitivos y pérdida de TAD estriatal.