Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the ß-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.

Moral Foundations Theory Among Autistic and Neurotypical Children.

Morality can help guide behavior and facilitate relationships. Although moral judgments by autistic people are similar to neurotypical individuals, many researchers argue that subtle differences signify deficits in autistic individuals. Moral foundation theory describes moral judgments in terms of differences rather than deficits. The current research, aimed at assessing autistic individuals' moral inclinations using Haidt's framework, was co-designed with autistic community members. Our aim was to describe autistic moral thinking from a strengths-based perspective while acknowledging differences that may pose interpersonal challenges among autistic youth. We assessed 25 autistic and 23 neurotypical children's moral judgments using the Moral Foundations Questionnaire for Kids. We used semi-structured interviews and qualitative analysis with a subset of participants to describe children's moral reasoning. Analyses suggested that autistic and neurotypical children make similar judgments about moral transgressions across all five moral foundations. General linear mixed modeling showed that the greatest predictor of recommending punishment was how bad children deemed moral transgressions to be. We also found a trend that autistic children were more likely to recommend punishment for harmless norms violations than were neurotypical children. Future research could use longitudinal methods to understand the development of moral judgments among autistic and neurotypical children.

Moral foundations theory in autism spectrum disorder: A qualitative investigation.

LAY ABSTRACT: Morality is important for how humans treat each other and non-human animals. Differences in moral thinking have been found between autistic and neurotypical individuals. This research has relied on ways of thinking about moral psychology that suggest that mature morals develop as individuals learn to take the perspectives of others. Yet, even autistic individuals, who sometimes differ in their ability to take others' perspectives, make moral judgements that are similar to neurotypical individuals. Moral foundations theory suggests that moral psychology is not hierarchical but differs depending on culture. This theory could therefore help make sense of similarities and differences in autistic and neurotypical moral thinking. Moral foundations theory has not yet been investigated among autistic individuals. In this study, we interviewed autistic adults as a first attempt at understanding how moral foundations theory fits with autistic moral thinking. We found that all five moral foundations of moral foundations theory were represented in the interviews, yet certain foundations appeared more prominent than others. The autistic adults interviewed in our study discussed issues of care and fairness more than of loyalty, authority or purity when prompted to discuss moral transgressions. Future research should use quantitative methods to compare groups of autistic and neurotypical individuals to clarify similarities and differences in moral thinking between the groups.

Psychometric Properties of the Merrill-Palmer-Revised Scales of Development in Preschool Children With Autism Spectrum Disorder.

Psychometrically sound tests of intellectual ability are indispensable for research and assessment of children with autism spectrum disorder (ASD), yet few tests have been validated for use with this population. The Merrill-Palmer-Revised Scales of Development (M-P-R) is a standardized test of intellectual ability that was validated for use with typically developing preschoolers. The current study's aim was to investigate the criterion validity of the M-P-R for assessing cognitive skills in preschoolers with ASD (N = 180). Good concurrent validity was demonstrated, with a large positive correlation between the M-P-R Receptive Language domain and the PLS-4 Auditory Comprehension subscale. The Cognitive domain of the M-P-R showed a medium positive correlation with later WISC-4 scores, showing acceptable predictive validity. Cognitive strengths and weaknesses assessed using the M-P-R mirrored those described for other measures, with most children obtaining higher standard scores on the Cognitive than the Receptive Language domain. An exploratory factor analysis suggested that one factor accounted for the majority of variability in M-P-R domains.

A novel working memory task for preschoolers: sensitivity to age differences from 3-5 years.

Working memory (WM) plays an important role in children's learning and is linked to later academic and occupational success. Understanding the early development of WM can provide critical clues regarding the underlying structure of executive functions and how they change over the life span. The main objectives of the present study were to (1) investigate age differences in the development of three components of WM (retrieval, substitution, transformation) on a novel preschool WM measure and (2) explore whether findings are consistent with the hierarchical model of WM development by examining perseverative and non-perseverative WM errors. Perseverative errors were hypothesized to be more strongly associated with problems substituting and transforming a representation held in mind, whereas non-perseverative errors were hypothesized to be associated with problems maintaining a representation in mind. Participants were 64 children ranging in age from 3.0 to 5.6 years. The results provide evidence for the sensitivity of the WM task to age differences from 3 to 5 years and support for the hierarchical model of WM development.