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Binge-eating disorder, the most prevalent eating disorder, is a serious public health problem associated with obesity, psychiatric and medical comorbidities, and functional impairments. Binge-eating disorder remains underrecognized and infrequently treated, and few evidence-based treatments exist. The authors tested the effectiveness of naltrexone-bupropion and behavioral weight loss therapy (BWL), alone and combined, for binge-eating disorder comorbid with obesity.In a randomized double-blind placebo-controlled trial conducted from February 2017 to February 2021, using a 2×2 balanced factorial design, 136 patients with binge-eating disorder (81.6% women; mean age, 46.5 years; mean BMI, 37.1) were randomized to one of four 16-week treatments: placebo (N=34), naltrexone-bupropion (N=32), BWL+placebo (N=35), or BWL+naltrexone-bupropion (N=35). Overall, 81.7% of participants completed independent posttreatment assessments.Intention-to-treat binge-eating remission rates were 17.7% in the placebo group, 31.3% in the naltrexone-bupropion group, 37.1% in the BWL+placebo group, and 57.1% in the BWL+naltrexone-bupropion group. Logistic regression of binge-eating remission revealed that BWL was significantly superior to no BWL, and that naltrexone-bupropion was significantly superior to placebo, but there was no significant interaction between BWL and medication. Mixed models of complementary measures of binge-eating frequency also indicated that BWL was significantly superior to no BWL. The rates of participants attaining 5% weight loss were 11.8% in the placebo group, 18.8% in the naltrexone-bupropion group, 31.4% in the BWL+placebo group, and 38.2% in the BWL+naltrexone-bupropion group. Logistic regression of 5% weight loss and mixed models of percent weight loss both revealed that BWL was significantly superior to no BWL. Mixed models revealed significantly greater improvements for BWL than no BWL on secondary measures (eating disorder psychopathology, depression, eating behaviors, and cholesterol and HbABWL and naltrexone-bupropion were associated with significant improvements in binge-eating disorder, with a consistent pattern of BWL being superior to no BWL.

The COVID-19 pandemic is a traumatic stressor resulting in anxiety, depression, post-traumatic stress, and burnout among healthcare workers. We describe an intervention to support the health workforce and summarize results from its 40-week implementation in a large, tri-state health system during the COVID-19 pandemic.We conducted 121 virtual and interactive Stress and Resilience Town Halls attended by 3555 healthcare workers. Town hall participants generated 1627 stressors and resilience strategies that we coded and analyzed using rigorous qualitative methods (Kappa = 0.85).We identify six types of stressors and eight types of resilience strategies reported by healthcare workers, how these changed over time, and how town halls were responsive to emerging health workforce needs. We show that town halls dedicated to groups working together yielded 84% higher mean attendance and more sharing of stressors and resilience strategies than those offered generally across the health system, and that specific stressors and strategies are reported consistently while others vary markedly over time.The virtual and interactive Stress and Resilience Town Hall is an accessible, scalable, and sustainable intervention to build mutual support, wellness, and resilience among healthcare workers and within hospitals and health systems responding to emerging crises, pandemics, and disasters.

BackgroundKetamine has emerged as one of the most promising therapies for treatment-resistant depression. However, inter-individual variability in response to ketamine is still not well understood and it is unclear how ketamine’s molecular mechanisms connect to its neural and behavioral effects.MethodsWe conducted a double-blind placebo-controlled study in which 40 healthy participants received acute ketamine (initial bolus 0.23 mg/kg, continuous infusion 0.58 mg/kg/hour). We quantified resting-state functional connectivity via data-driven global brain connectivity, related it to individual ketamine-induced symptom variation, and compared it to cortical gene expression targets.ResultsWe found that: i) both the neural and behavioral effects of acute ketamine are multi-dimensional, reflecting robust inter-individual variability; ii) ketamine’s data-driven principal neural gradient effect matched somatostatin (SST) and parvalbumin (PVALB) cortical gene expression patterns in humans, implicating the role of SST and PVALB interneurons in ketamine’s acute effects; and iii) behavioral data-driven individual symptom variation mapped onto distinct neural gradients of ketamine, which were resolvable at the single-subject level.ConclusionsCollectively, these findings support the possibility for developing individually precise pharmacological biomarkers for treatment selection in psychiatry.FundingThis study was supported by NIH grants DP5OD012109-01 (A.A.), 1U01MH121766 (A.A.), R01MH112746 (J.D.M.), 5R01MH112189 (A.A.), 5R01MH108590 (A.A.), NIAAA grant 2P50AA012870-11 (A.A.); NSF NeuroNex grant 2015276 (J.D.M.); Brain and Behavior Research Foundation Young Investigator Award (A.A.); SFARI Pilot Award (J.D.M., A.A.); Heffter Research Institute (Grant No. 1–190420); Swiss Neuromatrix Foundation (Grant No. 2016–0111m Grant No. 2015 – 010); Swiss National Science Foundation under the frame-work of Neuron Cofund (Grant No. 01EW1908), Usona Institute (2015 – 2056).

Aberrations of self-experience are considered a core feature of Borderline Personality Disorder (BPD). While prominent etiologic accounts of BPD, such as the mentalization based approach, appeal to the developmental constitution of self in early infant-caregiver environments, they often rely on a conception of self that is not explicitly articulated. Moreover, self-experience in BPD is often theorized at the level of narrative identity, thus minimizing the role of embodied experience. In this article, we present the hypothesis that disordered self and interpersonal functioning in BPD result, in part, from impairments in “embodied mentalization,” that manifest foundationally as alterations in minimal embodied selfhood, i.e. the first-person experience of being an individuated embodied subject. This account of BPD, which engages early intersubjective experiences has the potential to integrate phenomenological, developmental, and symptomatic findings in BPD, and is consistent with contemporary theories of brain function.

LEARNING OBJECTIVES After participating in this activity, learners should be better able to:• Assess medication management in patients with borderline personality disorder (BPD)• Evaluate the role of deprescribing as an active intervention in patients with BPD treated with polypharmacy ABSTRACT: Psychopharmacology in borderline personality disorder (BPD) is complicated by comorbid disorders, substance use, sensitivity to side effects, risk of self-harm through medication misuse, and intense but transient symptoms. Patients' relationships with medications may range from tenuous to highly enmeshed, and may profoundly influence the response to treatment. For these reasons, awareness of current evidence and flexible approaches are particularly relevant to prescribing in BPD. In this narrative review, we illustrate the current status of medication management in BPD by focusing on polypharmacy. We use a single vignette to explore the limitations of prescribing multiple medications and the factors contributing to polypharmacy. With the same vignette, and using the framework of deprescribing, we describe how medication regimens can be reduced to a necessary minimum. Deprescribing, originally developed in geriatric medicine, is an active intervention that involves a risk-benefit analysis for each medication, keeping in mind the patient's medical and psychiatric status and his or her preferences and values. Deprescribing lends itself well to use in psychiatry and especially in BPD because of its emphasis on the patient's preferences and on repeated conversations to revisit and update decisions. In addition to elaborating on the deprescribing framework, we provide recommendations for conducting these critical discussions about medications in BPD, with particular attention to the patient's relationship to the medication. Finally, we summarize our recommendations and strategies for implementing flexible and responsive medication management for patients with BPD. We suggest areas of future research, including testing the efficacy of targeted intermittent medication treatments.

Personal space (PS) regulation is a key component of effective social engagement. PS varies among individuals and is regulated by brain circuits involving the amygdala and the frontoparietal network. Others have reported that simulated PS intrusions suggest larger preferred interpersonal distance (PID) and a central role of amygdala hyperactivity in PS regulation in Borderline Personality Disorder (BPD). This study is the first report of live interpersonal distance preferences and relation to specific symptoms in BPD. We found a 2-fold larger PID in BPD than control (n=30, n=23). There were no significant differences in PID in BPD subject by medication status or pre-study diagnosis, and no significant correlations between PID and intensity of BPD, mood, anxiety, impulsive, or psychotic symptoms. In summary, PID is larger in BPD than control subjects. Unexpectedly, BPD subject PID did not differ in by medication status and did not correlate with intensity of any of the symptom types tested. We discuss these findings in context of severe attachment disturbances in BPD and the relationship between metaphoric social distance in the attachment framework. Future work is needed to identify neural circuits underlying PS regulation in BPD, individual differences in attachment, and relationship to symptom trajectory., Comment: pre-print

BackgroundVolatile interpersonal relationships are a core feature of Borderline Personality Disorder (BPD), and lead to devastating disruption of patients’ personal and professional lives. Quantitative models of social decision making and learning hold promise for defining the underlying mechanisms of this problem. In this study, we tested BPD and control subject weighting of social versus non-social information, and their learning about choices under stable and volatile conditions. We compared behavior using quantitative models.MethodsSubjects (n=20 BPD, n=23 control) played an extended reward learning task with a partner (confederate) that requires learning about non-social and social cue reward probability (The Social Valuation Task). Task experience was measured using language metrics: explicit emotions/beliefs, talk about the confederate, and implicit distress (using the previously established marker self-referentiality). Subjects’ weighting of social and non-social cues was tested in mixed-effects regression models. Subjects’ learning rates under stable and volatile conditions were modelled (Rescorla-Wagner approach) and group x condition interactions tested.ResultsCompared to controls, BPD subject debriefings included more mentions of the confederate and less distress language. BPD subjects also weighted social cues more heavily, but had blunted learning responses to (non-social and social) volatility.ConclusionsThis is the first report of patient behavior in the Social Valuation Task. The results suggest that BPD subjects expect higher volatility than do controls. These findings lay the groundwork for a neuro-computational dissection of social and non-social belief updating in BPD, which holds promise for the development of novel clinical interventions that more directly target pathophysiology.

Aberrations of self-experience are considered a core feature of Borderline Personality Disorder (BPD). While prominent etiologic accounts of BPD, such as the mentalization based approach, appeal to the developmental constitution of self in early infant-caregiver environments, they often rely on a conception of self that is not explicitly articulated. Moreover, self-experience in BPD is often theorized at the level of narrative identity, thus minimizing the role of embodied experience. In this article, we present the hypothesis that disordered self and interpersonal functioning in BPD result, in part, from impairments in “embodied mentalization,” that manifest foundationally as alterations in minimal embodied selfhood, i.e. the first-person experience of being an individuated embodied subject. This account of BPD, which engages early intersubjective experiences has the potential to integrate phenomenological, developmental, and symptomatic findings in BPD, and is consistent with contemporary theories of brain function.

Borderline personality disorder (BPD) is associated with deficits in neuropsychological measures such as memory, cognitive flexibility, and learning. Mixed findings suggest that deficits may be specific to particular aspects of learning. This is the first study to examine Kamin blocking in a BPD sample. A sample of 51 female subjects (N = 24 control, N = 27 BPD) were recruited. Participants completed a learning task in three phases: a first acquisition phase to learn cue-outcome associations, a second pairing phase to set up blocked cues, and a third testing phase to measure the extent of blocking and to test reversal learning. Participants indicated choice and certainty at each trial, and received feedback immediately after each trial. In phase one, mean correctness but not learning rate was less in BPD than control. Participants with BPD were also less certain about their responses, which correlated positively with correctness – this correlation was not found for control subjects. Kamin blocking, maintenance of previous learning, and reversal learning did not differ between groups. These results cohere with the idea that learning deficits in BPD are in specific domains, including the novel finding that Kamin blocking is preserved in BPD. The significant correlation between certainty and correctness in BPD may hint at mechanisms underlying the maintenance of low mood.

BackgroundOne aspect of selfhood that may have relevance for Borderline Personality Disorder (BPD) is variation in sense of body ownership. We employed the rubber hand illusion (RHI) to manipulate sense of body ownership in BPD. We extended previous research on illusory body ownership in BPD by testing: 1) two illusion conditions: asynchronous & synchronous stimulation, 2) relationship between Illusion experience and BPD symptoms, and 3) relationship between illusion experience and maladaptive personality traits.MethodsWe measured illusion strength (questionnaire responses), proprioceptive drift (perceived shift in physical hand position), BPD symptoms (DIB-R score), and maladaptive personality traits (PID-5) in 24 BPD and 21 control participants.ResultsFor subjective illusion strength, we found a main effect of group (BPD > HC, F = 11.94 p = 0.001), and condition (synchronous > asynchronous, F(1,43) = 22.80, p < 0.001). There was a group x condition interaction for proprioceptive drift (F(1,43) = 6.48, p = 0.015) such that people with BPD maintained illusion susceptibility in the asynchronous condition. Borderline symptom severity correlated with illusion strength within the BPD group, and this effect was specific to affective symptoms (r = 0.48, p < 0.01). Across all participants, trait psychoticism correlated with illusion strength (r = 0.44, p < 0.01).ConclusionPeople with BPD are more susceptible to illusory body ownership than controls. This is consistent with the clinical literature describing aberrant physical and emotional experience of self in BPD. A predictive-coding framework holds promise to develop testable mechanistic hypotheses for disrupted bodily self in BPD.HighlightsThe rubber hand illusion (RHI) allows measurement of self-disturbance.People with BPD had greater illusion susceptibility and this correlated with affective symptoms.Interoception stabilizes representations of body ownership, and is impaired in BPD.Illusion strength correlates with psychotic traits across levels of psychopathology.Predictive coding frameworks can probe mechanisms of impaired body ownership in psychopathology.

BackgroundLanguage use is of increasing interest in the study of mental illness. Analytical approaches range from phenomenological and qualitative to formal computational quantitative methods. Practically, the approach may have utility in predicting clinical outcomes. We harnessed a real-world sample (blog entries) from groups with psychosis, strong beliefs, odd beliefs, illness, mental illness and/or social isolation to validate and extend laboratory findings about lexical differences between psychosis and control subjects.MethodWe describe the results of two experiments using Linguistic Inquiry and Word Count software to assess word category frequencies. In experiment 1, we compared word use in psychosis and control subjects in the laboratory (23 per group), and related results to subject symptoms. In experiment 2, we examined lexical patterns in blog entries written by people with psychosis and eight comparison groups. In addition to between-group comparisons, we used factor analysis followed by clustering to discern the contributions of strong belief, odd belief and illness identity to lexical patterns.ResultsConsistent with others’ work, we found that first-person pronouns, biological process words and negative emotion words were more frequent in psychosis language. We tested lexical differences between bloggers with psychosis and multiple relevant comparison groups. Clustering analysis revealed that word use frequencies did not group individuals with strong or odd beliefs, but instead grouped individuals with any illness (mental or physical).ConclusionsPairing of laboratory and real-world samples reveals that lexical markers previously identified as specific language changes in depression and psychosis are probably markers of illness in general.

Objective Evaluation of liver transplant (LT) candidacy involves psychosocial evaluation to ensure appropriate organ allocation. However, the utility of pre-LT psychiatric and neuropsychological factors in predicting posttransplant outcomes remains uncertain. We reviewed current evidence on the prognostic value of pre-LT psychological factors for outcomes after LT. Method We conducted a systematic review of studies with adult LT recipients that investigate the relationship between pre-LT psychiatric and neuropsychological variables and posttransplant outcomes. We searched Ovid, MEDLINE, PsycINFO, EMBASE/Scopus, Cochrane Controlled trials register and Web of Science (January 1975 to May 2015) for longitudinal, peer-reviewed studies of at least 20 subjects and written in English. Results The 19 studies included in this review are heterogeneous in population, prognosis and duration of follow-up (from 20 days to more than 3 years). Findings on the prognostic value of pre-LT depression or anxiety on post-LT outcomes are mixed, though depression appears to predict lower quality of life (QOL). Pre-LT suicidal thoughts in particular are associated with post-LT depression. High submissiveness may predict rejection within 20 days of LT, and low conscientiousness is associated with greater nonadherence. Whereas pre-LT cognitive performance has not been shown to predict survival, poorer performance may predict poorer QOL after LT. Conclusion Further studies are needed to examine this important element of LT candidacy evaluation. Studies should evaluate psychiatric factors in large samples, include systematic evaluations by mental health clinicians and explore broader neuropsychological domains in predicting posttransplant outcomes.

Researchers investigating the psychological processes underlying specific mental health problems often have difficulties achieving large enough samples for adequately powered studies. This can be particularly problematic when studying psychopathology with low base rates in typical samples (i.e., undergraduate and community). A relatively new approach to recruitment and testing employs online crowdsourcing to rapidly measure the characteristics and behavior of large numbers of people. We tested the feasibility of researching borderline personality disorder (BPD) in this manner using one large crowdsourcing site, Amazon Mechanical Turk (MTurk). Specifically, we examined prevalence rates of psychopathology in a large MTurk sample, as well as the demographic, psychosocial, and psychiatric characteristics of individuals who met criteria for BPD. These characteristics were compared across three groups: those who met criteria for BPD currently, those who met criteria for remitted BPD, and those who had never met criteria for BPD. The results suggest that MTurk may be ideally suited for studying individuals with a wide range of pathology, from healthy to intensely symptomatic to remitted.

This chapter provides a summary of a landmark study on borderline personality disorder. What is the long-term prognosis of those with borderline personality disorder (BPD) compared to those with major depressive disorder or other personality disorders? Starting with that question, it describes the basics of the study, including funding, study location, who was studied, how many patients, study design, study intervention, follow-up, endpoints, results, and criticism and limitations. The findings show a favorable long-term prognosis that is comparable to that of other psychiatric disorders; 85% of those with BPS show at least 12-months of continuous remission after ten years. The chapter briefly reviews other relevant studies and information, discusses implications, and concludes with a relevant clinical case.

This chapter provides a summary of a landmark study on borderline personality disorder. Is dialectical behavior therapy more effective than treatment offered by nonbehavioral psychotherapy experts in reducing suicidal behaviors and treating borderline personality disorder? Starting with that question, it describes the basics of the study, including funding, study location, who was studied, how many patients, study design, study intervention, follow-up, endpoints, results, and criticism and limitations. The chapter briefly reviews other relevant studies and information, discusses implications, and concludes with a relevant clinical case.

This chapter provides a summary of a landmark study on borderline personality disorder. How does transference-focused psychotherapy, supportive therapy, and dialectical behavioral therapy compare in the treatment of borderline personality disorder? Starting with that question, the chapter describes the basics of this study, including funding sources, study location, who was studied, and how many patients participated in the study. The study design is described, as well as the study intervention, follow-up, endpoints, results, and finally a discussion of criticisms and limitations. The chapter briefly reviews other relevant studies and information, discusses implications, and concludes with a relevant clinical case.

BackgroundLanguage use is often disrupted in patients with schizophrenia; novel computational approaches may provide new insights.AimsTo test word use patterns as markers of the perceptual, cognitive and social experiences characteristic of schizophrenia.MethodWord counting software was applied to first-person accounts of schizophrenia and mood disorder.ResultsMore third-person plural pronouns (‘they’) and fewer first-person singular pronouns (‘I’) were used in schizophrenia than mood disorder accounts. Schizophrenia accounts included fewer words related to the body and ingestion, and more related to religion. Perceptual and causal language were negatively correlated in schizophrenia accounts but positively correlated in mood disorder accounts.ConclusionsDifferences in pronouns suggest decreased self-focus or perhaps even an understanding of self as other in schizophrenia. Differences in how perceptual and causal words are correlated suggest that long-held delusions represent a decreased coupling of explanations with sensory experience over time.

Purpose of review: We review the literature on the use and potential use of computational psychiatry methods in Borderline Personality Disorder. Recent findings: Computational approaches have been used in psychiatry to increase our understanding of the molecular, circuit, and behavioral basis of mental illness. This is of particular interest in BPD, where the collection of ecologically valid data, especially in interpersonal settings, is becoming more common and more often subject to quantification. Methods that test learning and memory in social contexts, collect data from real-world settings, and relate behavior to molecular and circuit networks are yielding data of particular interest. Summary: Research in BPD should focus on collaborative efforts to design and interpret experiments with direct relevance to core BPD symptoms and potential for translation to the clinic.

We reconsider delusions in terms of a "doxastic shear pin", a mechanism that errs so as to prevent the destruction of the machine (brain) and permit continued function (in an attenuated capacity). Delusions may disable flexible (but energetically expensive) inference. With each recall, delusions may be reinforced further and rendered resistant to contradiction. We aim to respond to deficit accounts of delusions - that delusions are only a problem without any benefit - by considering delusion formation and maintenance in terms of predictive coding. We posit that brains conform to a simple computational principle: to minimize prediction error (the mismatch between prior top-down expectation and current bottom-up input) across hierarchies of brain regions and psychological representation. Recent data suggest that delusions may form in the absence of constraining top-down expectations. Then, once formed, they become new priors that motivate other beliefs, perceptions, and actions by providing strong (sometimes overriding) top-down expectation. We argue that delusions form when the shear-pin breaks, permitting continued engagement with an overwhelming world, and ongoing function in the face of paralyzing difficulty. This crucial role should not be ignored when we treat delusions: we need to consider how a person will function in the world without them..

In mice, microRNAs (miRNAs) are required for embryonic viability, and previous reports implicate miRNA participation in brain cortical neurogenesis. Here, we provide a more comprehensive analysis of miRNA involvement in cortical brain development. To accomplish this we used mice in which Dicer, the RNase III enzyme necessary for canonical miRNA biogenesis, is depleted from Nestin-expressing progenitors and progeny cells. We systematically assessed how Dicer depletion impacts proliferation, cell death, migration and differentiation in the developing brain. Using markers for proliferation and in vivo labeling with thymidine analogs, we found reduced numbers of proliferating cells, and altered cell cycle kinetics from embryonic day 15.5 (E15.5). Progenitor cells were distributed aberrantly throughout the cortex rather than restricted to the ventricular and subventricular zones. Activated Caspase3 was elevated, reflecting increased cortical cell death as early as E15.5. Cajal–Retzius-positive cells were more numerous at E15.5 and were dysmorphic relative to control cortices. Consistent with this, Reelin levels were enhanced. Doublecortin and Rnd2 were also increased and showed altered distribution, supporting a strong regulatory role for miRNAs in both early and late neuronal migration. In addition, GFAP staining at E15.5 was more intense and disorganized throughout the cortex with Dicer depletion. These results significantly extend earlier works, and emphasize the impact of miRNAs on neural progenitor cell proliferation, apoptosis, migration, and differentiation in the developing mammalian brain.

The ocelli are three simple photoreceptors on the vertex of the fruit fly head. We sought to identify the gene encoded by the classical ocellar mutant, reduced ocelli (rdo). Deficiency and inversion breakpoint mapping and P-element induced male recombination analyses were performed and Pray For Elves (PFE; CG15151; Fbgn0032661) emerged as a promising candidate for the rdo phenotype. The PFE locus maps to polytene region 36E on chromosome 2L between elfless (Fbgn0032660) and Arrestin 1 (Fbgn0000120). FlyBase annotation predicts that PFE encodes a serine/threonine kinase, yet protein prediction programs revealed no kinase domain. These analyses suggest that PFE simply encodes a leucine rich repeat molecule of unknown function, but presumably functions in nervous system protein-protein interaction. Two classical spontaneous alleles of rdo, rdo(1) and rdo(2), were characterized and the underlying mutations result from a small deletion spanning exon 1/intron 1 and a B104/roo insertion into the 3'UTR of PFE, respectively. Transposase-mediated excisions of several P-elements inserted into the PFE locus revert the rdo phenotype and a full-length PFE cDNA is sufficient to rescue rdo. A Gal4 enhancer trap reveals a broad adult neural expression pattern for PFE. Our identification and initial characterization of the rdo locus will contribute to the understanding of neurogenesis and neural development in the simple photoreceptors of the Drosophila visual system.

MicroRNA (miRNA) function is required for normal animal development, in particular in differentiation pathways from stem cell and precursor populations. In neurogenesis, it is becoming increasingly appreciated that miRNAs act at many stages to ensure proper progression. In this study we examined the role of miR-34a in neural progenitor cells (NPC) derived from murine embryonic cortex. We found that over-expression of miR-34a in NPC significantly reduced the neuron yield upon in vitro induction of differentiation. MiR-34a has several predicted targets in the Notch pathway, which operates to balance progenitor self-renewal and differentiation during cortical neurogenesis. We tested several Notch pathway players for regulation by miR-34a in undifferentiated NPC, and found that mRNA and protein levels of Numbl, a negative regulator of Notch signaling, as well as two downstream pro-neural genes usually blocked by Notch signaling, NeuroD1 and Mash1, were diminished, while Notch1 and Cbf1 transcripts were enhanced by miR-34a over-expression. Using a luciferase reporter assay, we verified the Numbl 3′-UTR as a direct miR-34a target. Correspondingly, knock-down of endogenous miR-34a resulted in increased Numbl, NeuroD1 and Mash1, and reduced Notch1 transcript levels. Together these results implicate Numbl as a physiologically relevant target of miR-34a in NPC, allowing for enhanced Notch signaling and inhibition of neuronal differentiation. This work extends our understanding of miR-34a-mediated control of cell differentiation from cancer to mammalian nervous system development.

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3′ untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

RNA interference (RNAi) refers to post-transcriptional reduction of cellular gene expression mediated by sequence specific, 21 nucleotide double-stranded RNA molecules. RNAi occurs naturally in mammals (microRNAs), and appears to be particularly involved in controlling development of the nervous system. In addition, RNAi can be induced through exogenous expression of short hairpin RNAs (shRNA) using viral vectors. Feline immunodeficiency virus (FIV)- based lentiviral vectors are useful tools for neuronal gene delivery because they can stably infect difficult-to-transduce cells, are easy to produce, and, unlike the more broadly utilized HIV-based vectors, are non-human pathogens that pose a lower risk of generating replication-competent viruses in humans. Despite these advantages, compared to HIV vectors, less is known about the biology and construction of FIV and FIV-based vectors. Here, we describe thedevelopment of a second-generation FIV-based vector system that expresses U6 promoter-transcribed shRNAs (shLacZ) and a GFP reporter gene from the same vector. We found that the position of the U6 expression cassette with respect to the GFP reporter and viral rev-responsive element (RRE) was critical for reporter gene and shRNA expression. In particular, placement of the U6 promoter alone or an U6.shLacZ cassette near the 3' end of the vector genome between the GFP reporter cassette and the viral RRE caused 31 and 131-fold reductions, respectively, in transducing unit titer compared to an FIV vector expressing GFP alone. Reduced titer was also observed when a similarly-organized RNA pol III promoter (H1) was cloned into the same site. In contrast, TU titer was slightly increased (2.8-fold) when a similarly-sized, non-coding fragment of firefly luciferase was cloned into the same site downstream of GFP. Movement of U6.shLacZ to a site upstream of GFP resulted in complete TU titer restoration. Taken together, these data demonstrate that GFP expression is inhibited when a pol III-based promoter is located downstream of the reporter, suggesting that this inhibition may depend on recruitment of pol III transcription machinery.

MicroRNAs (miRNAs) are endogenously expressed noncoding RNAs that regulate mRNA expression. In vertebrates, more distinct miRNAs are expressed in the brain than in any other tissue, where they are hypothesized to function in neural development. Recent reports describing the effects of specific miRNAs during development, and studies employing miRNA depletion as neural commitment proceeds in the embryo, support a requisite role for miRNAs in cell-fate decisions and provide clues to their function in other aspects of nervous system development.