Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dosedependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications.

SHIELD: a platform for high-throughput screening of barrier-type DNA elements in human cells.

Chromatin boundary elements contribute to the partitioning of mammalian genomes into topological domains to regulate gene expression. Certain boundary elements are adopted as DNA insulators for safe and stable transgene expression in mammalian cells. These elements, however, are ill-defined and less characterized in the non-coding genome, partially due to the lack of a platform to readily evaluate boundary-associated activities of putative DNA sequences. Here we report SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains), a platform tailored for the high-throughput screening of barrier-type DNA elements in human cells. SHIELD takes advantage of the high specificity of serine integrase at heterochromatin, and exploits the natural heterochromatin spreading inside lamina-associated domains (LADs) for the discovery of potent barrier elements. We adopt SHIELD to evaluate the barrier activity of 1000 DNA elements in a high-throughput manner and identify 8 candidates with barrier activities comparable to the core region of cHS4 element in human HCT116 cells. We anticipate SHIELD could facilitate the discovery of novel barrier DNA elements from the non-coding genome in human cells.

Incorporation of noncanonical base Z yields modified mRNA with minimal immunogenicity and improved translational capacity in mammalian cells.

Chemically modified mRNAs hold great potential for therapeutic applications in vivo. Currently, the base modification scheme largely preserves the canonical Watson-Crick base pairing, thus missing one mode of mRNA modulation by altering its secondary structure. Here we report the incorporation of base Z (2-aminoadenine) into mRNA to create Z-mRNA with improved translational capacity, decreased cytotoxicity, and drastically reduced immunogenicity compared to the unmodified mRNA in mammalian cells. In particular, the A-to-Z substitution renders modified mRNAs less immunogenic than the state-of-the-art base modification N1-methylpseudouridine (m1ψ) in mouse embryonic fibroblast cells. As a proof of concept, we developed a Z-mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antigen-encoding Z-mRNA elicited substantial humoral and cellular immune responses in vivo in mice, albeit with relatively lower efficacy than the state-of-the-art m1ψ-mRNA. Z-mRNA expands the scope of mRNA base modifications toward noncanonical bases and could offer an advantageous platform for mRNA-based therapeutics where minimal immunogenicity is desired.

Lifetime psychiatric disorders in school-aged offspring of parents with bipolar disorder: the Pittsburgh Bipolar Offspring study.

CONTEXT: Whether offspring of parents with bipolar disorder (BP) are at specifically high risk to develop BP and other psychiatric disorders has not been adequately studied. OBJECTIVE: To evaluate lifetime prevalence and specificity of psychiatric disorders in offspring of parents with BP-I and BP-II. DESIGN: Offspring aged 6 to 18 years who have parents with BP and community control subjects were interviewed with standardized instruments. All research staff except the statistician were blind to parental diagnoses. SETTING: Parents with BP were recruited primarily through advertisement and outpatient clinics. Control parents were ascertained by random-digit dialing and were group matched for age, sex, and neighborhood to parents with BP. PARTICIPANTS: Three hundred eighty-eight offspring of 233 parents with BP and 251 offspring of 143 demographically matched control parents. MAIN OUTCOME MEASURES: Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) Axis I disorders. RESULTS: Adjusting for demographic factors, living with 1 vs both biological parents, both biological parents' non-BP psychopathology, and within-family correlations, offspring of parents with BP showed high risk for BP spectrum disorders (odds ratio [OR] = 13.4; 95% confidence interval [CI], 2.9-61.6) and any mood (OR = 5.2; 95% CI, 2.3-11.4), anxiety (OR = 2.3; 95% CI, 1.3-4.0), and Axis I (OR = 2.2; 95% CI, 1.5-3.3) disorders. Offspring of parents with BP with high socioeconomic status showed more disruptive behavior disorders and any Axis I disorders than offspring of control parents with high socioeconomic status. Families in which both parents had BP had more offspring with BP than families with only 1 parent with BP (OR = 3.6; 95% CI, 1.1-12.2). More than 75.0% of offspring who developed BP had their first mood episode before age 12 years, with most of these episodes meeting criteria for BP not otherwise specified and, to a lesser degree, major depression. CONCLUSIONS: Offspring of parents with BP are at high risk for psychiatric disorders and specifically for early-onset BP spectrum disorders. These findings further support the familiality and validity of BP in youth and indicate a need for early identification and treatment.

Replicable differences in preferred circadian phase between bipolar disorder patients and control individuals.

Morningness/eveningness (M/E) is a stable, quantifiable measure reflecting preferred circadian phase. Two prior studies suggest that bipolar I disorder (BP1) cases are more likely to have lower M/E scores, i.e., be evening types compared with control groups. These studies did not recruit controls systematically and did not evaluate key clinical variables. We sought to replicate the reported associations in a large, well defined sample, while evaluating potential confounding factors. Adults with bipolar disorder (BP) were compared with community controls drawn randomly from the same residential areas (190 cases and 128 controls). M/E was evaluated using the composite scale of morningness (CSM). After accounting for variables correlated with M/E, BP cases had significantly lower CSM scores than controls (i.e., more evening-type or fewer morning-type). There were no significant differences in M/E scores between BP1 or BP2 disorder cases (n=134 and 56, respectively). CSM scores were stable over approximately 2 years in a subgroup of participants (n=52). Individuals prescribed anxiolytic drugs, antidepressants, antipsychotic drugs, mood stabilizers or stimulant drugs had significantly lower age-corrected CSM scores compared with persons not taking these drugs. BP cases are more likely to be evening types, suggesting circadian phase delay in BP cases. Individuals with elevated depressive mood scores are more likely to be evening types. Our results suggest a replicable relationship between circadian phase and morbid mood states.

Schedule for affective disorders and schizophrenia for school-age children (K-SADS-PL) for the assessment of preschool children--a preliminary psychometric study.

OBJECTIVE: To assess the psychometrics of the schedule for affective disorders and schizophrenia for school-age children present and lifetime version (K-SADS-PL) in diagnosing DSM-IV psychiatric disorders and subsyndromal symptomatology in preschool children. METHOD: Parents were interviewed about their children using the K-SADS-PL, and they completed the early childhood inventory-4 (ECI-4) and child behavior checklist for ages 1(1/2)-5 years (CBCL). Discriminant, divergent, and convergent validity of the K-SADS-PL were evaluated in 204 offspring ages 2-5 years old of parents from an ongoing study. Inter-rater reliability as well as predictive validity of intake diagnoses at second assessment approximately two years after intake were evaluated. Fourteen children were also assessed by the preschool age psychiatric assessment (PAPA). RESULTS: Children who were diagnosed with oppositional defiant disorder, attention deficit hyperactivity disorder, anxiety, mood, or elimination disorders had significantly higher scores on the ECI-4 than children without these disorders. Significant correlations were found for all convergent CBCL scales. Divergent validity was acceptable for emotional disorders. Inter-rater kappa coefficients for all diagnoses were good. Above noted results were similar for children with at least one positive K-SADS-PL key screen symptom. A significantly higher percentage of children with an intake diagnosis had a diagnosis approximately two years after intake compared to those without an intake disorder. Overall, there was consistency between the PAPA and the K-SADS-PL. CONCLUSIONS: Pending further testing, the K-SADS-PL may prove useful for the assessment of psychopathology in preschoolers.

Mentoring increases connectedness and knowledge: a cross-sectional evaluation of two programs in child and adolescent psychiatry.

OBJECTIVE: The authors assess changes in knowledge and feeling connected to the field of child and adolescent psychiatry (CAP) after participation in a brief mentoring program held at two CAP conferences. METHODS: Similar mentorship programs were implemented at two CAP conferences, one national (N=119 participants), one international (N=53). The 4-day programs were part of larger travel awards, and included daily small group meetings consisting of a mode of two mentors and six participants. The authors created a survey with 40 quantitative questions designed to measure the change in participants' perceptions related to the conference and mentoring program, and provided additional fields for narrative comments. RESULTS: Mean participant ratings were positive for all questions on the survey. Changes in connectedness were rated higher than those in knowledge. The highest mean ratings were related to feeling more connected to the host organization, to CAP, and to other program participants. Outcomes were similar between the two conferences, except for knowledge gained on research, which was higher among participants in the international meeting. Outcomes were similar across demographic variables, except for internationally trained participants rating higher on research knowledge, connectedness, and overall knowledge. Over 75% of participants felt they made a connection with their mentor, bonded as a group, and learned new things about CAP and the host organization. A qualitative review of comments revealed several themes, including heightened importance of networking, increased awareness of the field, improved connectedness, a desire for trainee-focused events, and mixed feelings about how much structure to provide within the mentorship experience. CONCLUSION: A brief group-style mentoring program is logistically feasible within large conferences, and can result in broad positive impact for trainees. Future studies are warranted to determine if these programs have lasting effects on connectedness, career choice, and career development.

Mother-child interactions in depressed children and children at high risk and low risk for future depression.

OBJECTIVE: To compare mother-child interactions and parenting styles in families of children with major depressive disorder, youths at high risk for depression, and healthy controls. METHOD: Currently depressed (n = 43), high-risk (n = 28), and healthy control (n = 41) youths and their mothers engaged in a standardized videotaped problem-solving interaction. Measures of affect and behavior for both mothers and children were obtained, in addition to global measures of parenting. RESULTS: Depressed children demonstrated more negativity and less positivity in dyadic interactions than did children at high risk and control children. Mothers of depressed children were more disengaged than control mothers. Exploratory repeated-measures analyses in a subgroup of depressed children (n = 16) suggested mother-child interactions do not significantly change when children recover from depression. Children at high risk demonstrated less positivity in dyadic interactions than did controls. Mothers with a history of major depressive disorder and mothers with higher current depressive symptoms demonstrated patterns of disengagement and low control in interactions with children. CONCLUSIONS: Mother-child interactions in depressed youths are marked by maternal disengagement and low child positivity that may not improve when children recover. The bidirectional effects of maternal disengagement and low levels of child positivity may precede onset of major depressive disorder in children and serve as risk factors for recurrent depression in youths.

Aggression, hostility, and irritability in children at risk for bipolar disorder.

OBJECTIVES: To assess aggression, irritability and hostility in children at risk for bipolar disorder (BP). METHODS: Using the parent and the child versions of the Children's Hostility Inventory (CHI), we assessed aggression, hostility, and irritability in 300 offspring aged 6-18 years old of BP parents and 169 children of community controls. RESULTS: Children of BP parents have significantly higher scores on the total CHI and its subscales than do children of control parents. After adjusting for demographic variables, both parents' non-BP psychopathology, child psychopathology, and within-family correlations, three factors remain significant: total CHI by parent rating, irritability subscale by parent rating, and irritability by child self-report. The hostility subscale by parent rating became a trend. CONCLUSIONS: Children of BP parents score higher on ratings of hostility and irritability than children of community control parents, independent of child psychopathology and non-BP parental psychopathology. Follow-up of these children to evaluate whether these symptoms are markers for the development of BP or mood disorders is warranted.

Fluoxetine for the treatment of childhood anxiety disorders: open-label, long-term extension to a controlled trial.

OBJECTIVE: To assess the efficacy of fluoxetine for the long-term treatment of children and adolescents with anxiety disorders, including generalized anxiety disorder, separation anxiety disorder, and/or social phobia. METHOD: Children and adolescents (7-17 years old) with anxiety disorders were studied in open treatment for 1 year after they completed a randomized, controlled trial (RCT) comparing fluoxetine and placebo. The follow-up phase assessments included clinician, parent, and child ratings with measures of global severity, global improvement, and anxiety symptoms. RESULTS: Subjects taking fluoxetine (n = 42) were compared with those taking no medication (n = 10) during follow-up on anxiety changes from the end of the RCT through the follow-up period. Statistical models included RCT assignment and follow-up psychological treatment. Excluded subjects took other medications (n = 4) or did not complete follow-up (n = 18). Compared with subjects taking no medication, subjects taking fluoxetine showed significantly superior follow-up outcomes on most measures, including clinician, parent, and child ratings. CONCLUSIONS: The results suggest that fluoxetine is clinically effective for the maintenance treatment of anxiety disorders in children and adolescents. A major limitation, however, was the lack of RCT methodology in the follow-up phase. RCTs are needed to determine the long-term risks and benefits of fluoxetine for this group.

Citalopram treatment of pediatric recurrent abdominal pain and comorbid internalizing disorders: an exploratory study.

OBJECTIVE: To assess the potential efficacy, tolerability, and safety of citalopram in the treatment of functional pediatric recurrent abdominal pain and comorbid internalizing disorders. METHOD: Twenty-five clinically referred children and adolescents with recurrent abdominal pain aged 7 to 18 years, inclusive, participated in a 12-week, flexible-dose, open-label trial of citalopram. Primary outcome measure was the Clinical Global Impression Scale-Improvement, with responders defined by ratings of 1 (very much improved) or 2 (much improved). Secondary measures included self- and parent reports of abdominal pain, anxiety, depression, other somatic symptoms, and functional impairment. Side effects were assessed using a standardized checklist. Data were analyzed using an intent-to-treat format and the last observation carried forward procedure. RESULTS: Twenty-one subjects (84%) were classified as responders (Clinical Global Impression Scale-Improvement score < or =2). Citalopram was generally well tolerated. Four subjects withdrew prematurely, one due to reported visual side effects. Ratings of abdominal pain, anxiety, depression, other somatic symptoms, and functional impairment all improved significantly over the course of the study compared with baseline. CONCLUSIONS: Citalopram is a promising treatment for functional pediatric recurrent abdominal pain and deserves additional study with a randomized, placebo-controlled clinical trial.

Recurrent abdominal pain, anxiety, and depression in primary care.

OBJECTIVE: The prevalence of psychiatric disorder in children and adolescents with functional recurrent abdominal pain (RAP) is unknown. Our aim was to determine whether RAP is associated with psychiatric symptoms and disorders, anxious temperament, and functional impairment in pediatric primary care. METHODS: Children and adolescents who were 8 to 15 years of age, inclusive, and presented with RAP (N = 42) or for routine care in the absence of recurrent pain (N = 38) were identified by a screening procedure in pediatric primary care office waiting rooms and recruited to participate in a case-control study. Outcome measures were psychiatric diagnoses generated by standardized psychiatric interview administered blind to subject status and self, parent, and clinician ratings of child psychiatric symptoms, temperamental traits, and functional status. RESULTS: RAP patients were significantly more likely to receive a diagnosis of a psychiatric disorder, with a categorical anxiety disorder in 33 (79%) and a depressive disorder in 18 patients (43%), and higher levels of anxiety and depressive symptoms, temperamental harm avoidance, and functional impairment than control subjects. Anxiety disorders (mean age of onset: 6.25 [standard deviation: 2.17] years) were significantly more likely to precede RAP (mean age of onset: 9.17 [standard deviation: 2.75] years) in patients with associated anxiety. CONCLUSIONS: Youths who present with RAP in primary care deserve careful assessment for anxiety and depressive disorders. Future studies should examine treatments that are proved to be efficacious for pediatric anxiety and/or depressive disorders as potential interventions for RAP. Longitudinal, family, and psychobiological studies are needed to illuminate the nature of observed associations among RAP, anxiety, and depression.

Fluoxetine for the treatment of childhood anxiety disorders.

OBJECTIVE: To assess the efficacy and tolerability of fluoxetine for the acute treatment of children and adolescents with generalized anxiety disorder, separation anxiety disorder, and/or social phobia. METHOD: Anxious youths (7-17 years old) who had significant functional impairment were randomized to fluoxetine (20 mg/day) (n = 37) or placebo (n = 37) for 12 weeks. RESULTS: Fluoxetine was effective in reducing the anxiety symptoms and improving functioning in all measures. Using intent-to-treat analysis, 61% of patients taking fluoxetine and 35% taking placebo showed much to very much improvement. Despite this improvement, a substantial group of patients remained symptomatic. Fluoxetine was well tolerated except for mild and transient headaches and gastrointestinal side effects. Youths with social phobia and generalized anxiety disorder responded better to fluoxetine than placebo, but only social phobia moderated the clinical and functional response. Severity of the anxiety at intake and positive family history for anxiety predicted poorer functioning at the end of the study. CONCLUSIONS: Fluoxetine is useful and well tolerated for the acute treatment of anxious youths. Investigations regarding the optimization of treatment to obtain full anxiety remission and the length of treatment necessary to prevent recurrences are warranted.

Is bipolar disorder specifically associated with panic disorder in youths?

OBJECTIVE: To replicate previous findings of high rates of bipolar disorder (BPD) in patients with panic disorder (PD) and determine if youths with both PD and BPD have more severe illness. METHOD: 2025 youths aged 5 to 19 years seen at a mood and anxiety specialty clinic were assessed using the Schedule for Affective Disorders and Schizophrenia for School Aged Children-Present Episode, 4th Revision. Diagnoses were made using DSM-III and DSM-III-R criteria and then updated to conform to DSM-IV criteria. Patients were grouped into those with PD (N = 42), those with non-PD anxiety disorders (N = 407), and psychiatric controls with no anxiety diagnosis (N = 1576). RESULTS: Youths with PD were more likely to exhibit comorbid BPD (N = 8, 19.0%) than youths with either non-PD anxiety disorders (N = 22, 5.4%) or other nonanxious psychiatric disorders (N = 112, 7.1%). The symptoms of PD and mania were not affected by the comorbidity between PD and BPD. Youths with both PD and BPD had more psychotic symptoms and suicidal ideation than patients with PD and other non-bipolar psychiatric disorders and BPD patients with other nonanxious comorbid disorders. CONCLUSION: The presence of either PD or BPD in youths made the co-occurrence of the other condition more likely, as has been noted in adults. Patients with both PD and BPD are more likely to have psychotic symptoms and suicidal ideation. In treating youths with PD, clinicians must be vigilant for possible comorbid BPD or risk of pharmacologic triggering of a manic or hypomanic episode. Prospective studies are needed to learn if PD predicts the onset of BPD in children and adolescents.