The functional impact of 1,570 individual amino acid substitutions in human OTC.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Individuals with neonatal onset appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma, and death, outcomes ameliorated by rapid diagnosis and treatment. Here, we develop a high-throughput functional assay for human OTC and individually measure the impact of 1,570 variants, 84% of all SNV-accessible missense mutations. Comparison to existing clinical significance calls, demonstrated that our assay distinguishes known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. This functional stratification allowed us to identify score ranges corresponding to clinically relevant levels of impairment of OTC activity. Examining the results of our assay in the context of protein structure further allowed us to identify a 13 amino acid domain, the SMG loop, whose function appears to be required in human cells but not in yeast. Finally, inclusion of our data as PS3 evidence under the current ACMG guidelines, in a pilot reclassification of 34 variants with complete loss of activity, would change the classification of 22 from variants of unknown significance to clinically actionable likely pathogenic variants. These results illustrate how large-scale functional assays are especially powerful when applied to rare genetic diseases.

Constructing and interpreting a large-scale variant effect map for an ultrarare disease gene: Comprehensive prediction of the functional impact of PSAT1 genotypes.

Reduced activity of the enzymes encoded by PHGDH, PSAT1, and PSPH causes a set of ultrarare, autosomal recessive diseases known as serine biosynthesis defects. These diseases present in a broad phenotypic spectrum: at the severe end is Neu-Laxova syndrome, in the intermediate range are infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end is childhood disease with intellectual disability. However, L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms. Therefore, knowledge of pathogenic variants can improve clinical outcomes. Here, we use a yeast-based assay to individually measure the functional impact of 1,914 SNV-accessible amino acid substitutions in PSAT. Results of our assay agree well with clinical interpretations and protein structure-function relationships, supporting the inclusion of our data as functional evidence as part of the ACMG variant interpretation guidelines. We use existing ClinVar variants, disease alleles reported in the literature and variants present as homozygotes in the primAD database to define assay ranges that could aid clinical variant interpretation for up to 98% of the tested variants. In addition to measuring the functional impact of individual variants in yeast haploid cells, we also assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. Results from our diploid assay successfully distinguish the genotypes of affected individuals from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements to predict the biallelic function of ~1.8 million allele combinations corresponding to potential human genotypes. Taken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of ultrarare diseases.

The antioxidant response favors Leishmania parasites survival, limits inflammation and reprograms the host cell metabolism.

The oxidative burst generated by the host immune system can restrict intracellular parasite entry and growth. While this burst leads to the induction of antioxidative enzymes, the molecular mechanisms and the consequences of this counter-response on the life of intracellular human parasites are largely unknown. The transcription factor NF-E2-related factor (NRF2) could be a key mediator of antioxidant signaling during infection due to the entry of parasites. Here, we showed that NRF2 was strongly upregulated in infection with the human Leishmania protozoan parasites, its activation was dependent on a NADPH oxidase 2 (NOX2) and SRC family of protein tyrosine kinases (SFKs) signaling pathway and it reprogrammed host cell metabolism. In inflammatory leishmaniasis caused by a viral endosymbiont inducing TNF-α in chronic leishmaniasis, NRF2 activation promoted parasite persistence but limited TNF-α production and tissue destruction. These data provided evidence of the dual role of NRF2 in protecting both the invading pathogen from reactive oxygen species and the host from an excess of the TNF-α destructive pro-inflammatory cytokine.

Whole genome sequencing of experimental hybrids supports meiosis-like sexual recombination in Leishmania.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.

Modeling CAR T-Cell Therapy with Patient Preconditioning.

The Federal Drug Administration approved the first Chimeric Antigen Receptor T-cell (CAR T-cell) therapies for the treatment of several blood cancers in 2017, and efforts are underway to broaden CAR T technology to address other cancer types. Standard treatment protocols incorporate a preconditioning regimen of lymphodepleting chemotherapy prior to CAR T-cell infusion. However, the connection between preconditioning regimens and patient outcomes is still not fully understood. Optimizing patient preconditioning plans and reducing the CAR T-cell dose necessary for achieving remission could make therapy safer. In this paper, we test treatment regimens consisting of sequential administration of chemotherapy and CAR T-cell therapy on a system of differential equations that models the tumor-immune interaction. We use numerical simulations of treatment plans from within the scope of current medical practice to assess the effect of preconditioning plans on the success of CAR T-cell therapy. Model results affirm clinical observations that preconditioning can be crucial for most patients, not just to reduce side effects, but to even achieve remission at all. We demonstrate that preconditioning plans using the same CAR T-cell dose and the same total concentration of chemotherapy can lead to different patient outcomes due to different delivery schedules. Results from sensitivity analysis of the model parameters suggest that making small improvements in the effectiveness of CAR T-cells in attacking cancer cells will significantly reduce the minimum dose required for successful treatment. Our modeling framework represents a starting point for evaluating the efficacy of patient preconditioning in the context of CAR T-cell therapy.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

Immunomodulatory and Antileishmanial Activity of Phenylpropanoid Dimers Isolated from Nectandra leucantha.

Three phenylpropanoid dimers (1-3) including two new metabolites were isolated from the extract of the twigs of Nectandra leucantha using antileishmanial bioassay-guided fractionation. The in vitro antiparasitic activity of the isolated compounds against Leishmania donovani parasites and mammalian cytotoxicity and immunomodulatory effects were evaluated. Compounds 1-3 were effective against the intracellular amastigotes within macrophages, with IC50 values of 26.7, 17.8, and 101.9 µM, respectively. The mammalian cytotoxicity, given by the 50% cytotoxic concentration (CC50), was evaluated against peritoneal macrophages. Compounds 1 and 3 were not toxic up to 290 µM, whereas compound 2 demonstrated a CC50 value of 111.2 µM. Compounds 1-3 also suppressed production of disease exacerbatory cytokines IL-6 and IL-10 but had minimal effect on nitric oxide production in L. donovani-infected macrophages, indicating that antileishmanial activity of these compounds is mediated via an NO-independent mechanism. Therefore, these new natural products could represent promising scaffolds for drug design studies for leishmaniasis.

The structure and repertoire of small interfering RNAs in Leishmania (Viannia) braziliensis reveal diversification in the trypanosomatid RNAi pathway.

Among trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in-depth analysis of small interfering RNAs (siRNAs) associated with L. braziliensis Argonaute1 (LbrAGO1). In contrast to T. brucei, Leishmania siRNAs are sensitive to 3' end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2'-O-methyltransferase, which modifies small RNA 3' ends. Consistent with this observation, ~20% of siRNA 3' ends carry non-templated uridines. Thus siRNA biogenesis, and most likely their metabolism, is different in these organisms. Similarly to T. brucei, putative mobile elements and repeats constitute the major Leishmania siRNA-producing loci and AGO1 ablation leads to accumulation of long transcripts derived from putative mobile elements. However, contrary to T. brucei, no siRNAs were detected from other genomic regions with the potential to form double-stranded RNA, namely sites of convergent transcription and inverted repeats. Thus, our results indicate that organism-specific diversification has occurred in the RNAi pathway during evolution of the trypanosomatid lineage.

Spatiotemporal dynamics of tumor - CAR T-cell interaction following local administration in solid cancers.

The success of chimeric antigen receptor (CAR) T-cell therapy in treating hematologic malignancies has generated widespread interest in translating this technology to solid cancers. However, issues like tumor infiltration, the immunosuppressive tumor microenvironment, and tumor heterogeneity limit its efficacy in the solid tumor setting. Recent experimental and clinical studies propose local administration directly into the tumor or at the tumor site to increase CAR T-cell infiltration and improve treatment outcomes. Characteristics of the types of solid tumors that may be the most receptive to this treatment approach remain unclear. In this work, we develop a spatiotemporal model for CAR T-cell treatment of solid tumors, and use numerical simulations to compare the effect of introducing CAR T cells via intratumoral injection versus intracavitary administration in diverse cancer types. We demonstrate that the model can recapitulate tumor and CAR T-cell data from imaging studies of local administration of CAR T cells in mouse models. Our results suggest that locally administered CAR T cells will be most successful against slowly proliferating, highly diffusive tumors, which have the lowest average tumor cell density. These findings affirm the clinical observation that CAR T cells will not perform equally across different types of solid tumors, and suggest that measuring tumor density may be helpful when considering the feasibility of CAR T-cell therapy and planning dosages for a particular patient. We additionally find that local delivery of CAR T cells can result in deep tumor responses, provided that the initial CAR T-cell dose does not contain a significant fraction of exhausted cells.

Heterogeneous SARS-CoV-2 kinetics due to variable timing and intensity of immune responses.

The viral kinetics of documented SARS-CoV-2 infections exhibit a high degree of inter-individual variability. We identified six distinct viral shedding patterns, which differed according to peak viral load, duration, expansion rate and clearance rate, by clustering data from 768 infections in the National Basketball Association cohort. Omicron variant infections in previously vaccinated individuals generally led to lower cumulative shedding levels of SARS-CoV-2 than other scenarios. We then developed a mechanistic mathematical model that recapitulated 1510 observed viral trajectories, including viral rebound and cases of reinfection. Lower peak viral loads were explained by a more rapid and sustained transition of susceptible cells to a refractory state during infection, as well as an earlier and more potent late, cytolytic immune response. Our results suggest that viral elimination occurs more rapidly during omicron infection, following vaccination, and following re-infection due to enhanced innate and acquired immune responses. Because viral load has been linked with COVID-19 severity and transmission risk, our model provides a framework for understanding the wide range of observed SARS-CoV-2 infection outcomes.

Heterogeneous SARS-CoV-2 kinetics due to variable timing and intensity of immune responses.

The viral kinetics of documented SARS-CoV-2 infections exhibit a high degree of interindividual variability. We identified 6 distinct viral shedding patterns, which differed according to peak viral load, duration, expansion rate, and clearance rate, by clustering data from 768 infections in the National Basketball Association cohort. Omicron variant infections in previously vaccinated individuals generally led to lower cumulative shedding levels of SARS-CoV-2 than other scenarios. We then developed a mechanistic mathematical model that recapitulated 1,510 observed viral trajectories, including viral rebound and cases of reinfection. Lower peak viral loads were explained by a more rapid and sustained transition of susceptible cells to a refractory state during infection as well as by an earlier and more potent late, cytolytic immune response. Our results suggest that viral elimination occurs more rapidly during Omicron infection, following vaccination, and following reinfection due to enhanced innate and acquired immune responses. Because viral load has been linked with COVID-19 severity and transmission risk, our model provides a framework for understanding the wide range of observed SARS-CoV-2 infection outcomes.

A unifying model to explain frequent SARS-CoV-2 rebound after nirmatrelvir treatment and limited prophylactic efficacy.

In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We develop a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulates viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly.

A unifying model to explain high nirmatrelvir therapeutic efficacy against SARS-CoV-2, despite low post-exposure prophylaxis efficacy and frequent viral rebound.

In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We developed a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulated viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly.

Predicting the functional effect of compound heterozygous genotypes from large scale variant effect maps.

Background: Pathogenic variants in PHGDH, PSAT1 , and PSPH cause a set of rare, autosomal recessive diseases known as serine biosynthesis defects. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately in the form of infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, as childhood disease with intellectual disability. However, because L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is highly actionable. Methods: Recently, our laboratory established a yeast-based assay for human PSAT1 function. We have now applied it at scale to assay the functional impact of 1,914 SNV-accessible amino acid substitutions. In addition to assaying the functional impact of individual variants in yeast haploid cells, we can assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. Results: Results of our assays of individual variants (in haploid yeast cells) agree well with clinical interpretations and protein structure-function relationships, supporting the use of our data as functional evidence under the ACMG interpretation guidelines. Results from our diploid assay successfully distinguish patient genotypes from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements (in haploid yeast cells) to accurately predict the biallelic function (in diploid yeast cells) of ~ 1.8 million allele combinations corresponding to potential human genotypes. Conclusions: Taken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of a rare disease.

Retention and loss of RNA interference pathways in trypanosomatid protozoans.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

Leishmanicidal activity of two naphthoquinones against Leishmania donovani.

Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (TR 001) and 2,3-dibromo-1,4-naphthoquinone (TR 002). TR 001 was more efficient than TR 002 in inducing killing of promastigotes and intracellular amastigotes. These values compare well to those obtained with the standard first-line antileishmanial agent sodium stibogluconate (SSG). TR 001 also induced significantly more nitric oxide (NO) production than TR 002 or SSG. Taken together, these data show that TR 001 and TR 002 could be promising new drugs for treatment of visceral leishmaniasis.

Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins.

Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence.

Understanding Client Difficulties in Transdiagnostic Internet-Delivered Cognitive Behaviour Therapy: A Qualitative Analysis of Homework Reflections.

Internet-delivered cognitive behaviour therapy (ICBT) is helpful for many clients, but less is known about the challenges clients face during ICBT, such as difficulties with skill practice, development, or maintenance. Understanding client difficulties can help therapists support clients with skill development and prevent treatment drop-out, but has not been systematically studied. This study included a conventional content analysis of clients' responses to a homework reflection question about difficulties with lessons and skills. Data was drawn from a previously published trial of 301 clients who were randomly assigned to receive homework reflection questions during ICBT. A decreasing number of clients responded to the question about skill difficulties with each lesson. Clients who answered the question about difficulties were more engaged with ICBT (i.e., more lessons completed, logins, days enrolled in ICBT, and messages sent to therapists). Clients shared skill-specific challenges (including initial challenges and more advanced challenges), generic challenges (content or skills being cognitively draining or emotionally draining, contextual challenges, forgetfulness, limited time, and lack of familiarity with the skill), or no challenges. Thought challenging (59.6%) and graded exposure (57.5%) were associated with the greatest number of skill-specific challenges. Findings can help therapists anticipate and address common client challenges during ICBT.