Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells ...remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.
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•Efficient ablation of B2M and CCR5 in human hematopoietic cells using CRISPR/Cas9•CRISPR/Cas9 CCR5-deleted CD34+ HSPCs retain multilineage engraftment potential•Minimal off-target mutational events in CD34+ HSPCs after CRISPR/Cas9 treatment
Genome editing has the potential to revolutionize cell-based gene therapy. In this study, Mandal et al. developed approaches for using CRISPR/Cas9 in human CD34+ HSPCs that yielded high on-target with minimal off-target mutagenesis. These results indicate that CRISPR/Cas9 could broadly enable gene and cell-based therapies of blood.
Few individuals who experience trauma develop posttraumatic stress disorder (PTSD). Therefore, the identification of individual differences that signal increased risk for PTSD is important. Lissek et ...al. (
2006
) proposed using a weak rather than a strong situation to identify individual differences. A weak situation involves less-salient cues as well as some degree of uncertainty, which reveal individual differences. A strong situation involves salient cues with little uncertainty, which produce consistently strong responses. Results from fear conditioning studies that support this hypothesis are discussed briefly. This review focuses on recent findings from three learning tasks: classical eyeblink conditioning, avoidance learning, and a computer-based task. These tasks are interpreted as weaker learning situations in that they involve some degree of uncertainty. Individual differences in learning based on behavioral inhibition, which is a risk factor for PTSD, are explored. Specifically, behaviorally inhibited individuals and rodents (i.e., Wistar Kyoto rats), as well as individuals expressing PTSD symptoms, exhibit enhanced eyeblink conditioning. Behaviorally inhibited rodents also demonstrate enhanced avoidance responding (i.e., lever pressing). Both enhanced eyeblink conditioning and avoidance are most evident with schedules of partial reinforcement. Behaviorally inhibited individuals also performed better on reward and punishment trials than noninhibited controls in a probabilistic category learning task. Overall, the use of weaker situations with uncertain relationships may be more ecologically valid than learning tasks in which the aversive event occurs on every trial and may provide more sensitivity for identifying individual differences in learning for those at risk for, or expressing, PTSD symptoms.
Individuals differ in how they react to stress or trauma through different coping styles in which they may deal directly with a stressor by adopting approach coping styles or disengage with a ...stressor by utilizing avoidant coping styles. Avoidant coping styles have been linked to adverse outcomes including psychological distress, anxiety disorders, and post-traumatic stress disorder (PTSD). Recently, avoidance coping styles as measured by a subset of items on the Brief COPE were found to have a weak positive relationship with performance on a computer-based avatar task which is related to avoidant personality temperaments. This avatar task was developed as an alternative for paper and pencil self-report inventories for measuring avoidant tendencies based on possible response biases of avoidant individuals. In the current study, avoidance and approach coping styles as measured by the Brief Approach/Avoidance Coping Questionnaire (BACQ) were compared to avoidant coping as measured by the Brief COPE and performance on the avatar task. In addition to approach and avoidance coping, the BACQ also measures active avoidance coping (i.e., diversion) and passive avoidance coping (i.e., resignation and withdrawal). The relationships between approach and avoidance coping and performance on the avatar task were also analyzed with the outcome of perceived stress as measured by the Perceived Stress Scale (PSS).
One hundred undergraduates voluntarily completed the BACQ, the Brief COPE, and the PSS. Participants also completed a computer-based task in which they guided an avatar through a series of social situations where they indicated how they would interact with or avoid interacting with strangers.
Approach coping had a weak negative relationship to avoidance coping as measured by the BACQ and the Brief COPE. Performance on the avatar task had a moderate positive relationship with avoidance coping (diversion as well as resignation and withdrawal) as measured by the BACQ and a moderate negative relationship with approach coping as measured by the BACQ. A model including only approach, diversion, and resignation and withdrawal coping best predicted performance on the avatar task in a linear regression model. While resignation and withdrawal coping and diversion coping had moderate positive relationships to avatar task scores, only resignation and withdrawal had a strong positive relationship to perceived stress. A model than included only resignation and withdrawal coping best predicted perceived stress in a linear regression model. Overall, passive avoidant coping styles (i.e., resignation and withdrawal), but not active avoidant coping style (i.e., diversion), were related to perceived stress. These results support the continued study of multiple aspects of avoidant coping styles as well as the avatar task to increase our understanding of the maladaptive effects of excessive avoidance in the face of stress.
An estimated 34 million people are living with HIV worldwide (UNAIDS, 2012), with the number of infected persons rising every year. Increases in HIV prevalence have resulted not only from new ...infections, but also from increases in the survival of HIV-infected persons produced by effective anti-retroviral therapies. Augmentation of anti-viral immune responses may be able to further increase the survival of HIV-infected persons. One strategy to augment these responses is to reinvigorate exhausted anti-HIV immune cells present in chronically infected persons. The PD-1-PD-L1 pathway has been implicated in the exhaustion of virus-specific T cells during chronic HIV infection. Inhibition of PD-1 signaling using blocking anti-PD-1 antibodies has been shown to reduce simian immunodeficiency virus (SIV) loads in monkeys. We now show that PD-1 blockade can improve control of HIV replication in vivo in an animal model. BLT (Bone marrow-Liver-Thymus) humanized mice chronically infected with HIV-1 were treated with an anti-PD-1 antibody over a 10-day period. The PD-1 blockade resulted in a very significant 45-fold reduction in HIV viral loads in humanized mice with high CD8(+) T cell expression of PD-1, compared to controls at 4 weeks post-treatment. The anti-PD-1 antibody treatment also resulted in a significant increase in CD8(+) T cells. PD-1 blockade did not affect T cell expression of other inhibitory receptors co-expressed with PD-1, including CD244, CD160 and LAG-3, and did not appear to affect virus-specific humoral immune responses. These data demonstrate that inhibiting PD-1 signaling can reduce HIV viral loads in vivo in the humanized BLT mouse model, suggesting that blockade of the PD-1-PD-L1 pathway may have therapeutic potential in the treatment of patients already infected with the AIDS virus.
HIV needs to be fit to transmitAlthough you might not think it, it's hard to catch HIV. Less than 1% of unprotected sexual exposures result in infection. What then leads to transmission? Carlson et ...al. determined the amino acid sequence of viruses infecting 137 Zambian heterosexual couples in which one partner infected the other (see the Perspective by Joseph and Swanstrom). The authors then used statistical modeling and found that transmitted viruses are typically the most evolutionarily fit. That is, compared to other viral variants in the infected person, the transmitted virus most closely matches the most common viral sequence found in the Zambian population.Science, this issue 10.1126/science.1254031; see also p. 136
Current psychotherapies seek to reduce experiential avoidance (EA) which has also been put forth as a risk factor for anxiety disorders, depression, and post-traumatic stress disorder. EA is a ...potentially maladaptive self-regulatory tendency to avoid negative thoughts, feelings, memories, physical sensations, and other internal experiences. One unresolved issue with the most commonly used measures of EA, the Acceptance and Action Questionnaire-II (AAQ-II) which measures EA as a single factor and the Multidimensional Experiential Avoidance Questionnaire (MEAQ) which measures EA as six subdimensions, is what exactly is being measured. The AAQ-II appears to measure negative affect (NA), some aspects of avoidant coping, and psychological distress. In addition, the relationships of all the MEAQ subscales have not been thoroughly examined with these other constructs. In the current study, the relationships of AAQ-II and MEAQ scores with NA, avoidant coping styles, and perceived stress were examined.
Two-hundred undergraduates (154 females and 46 males) completed the AAQ-II and MEAQ, the Distressed Type D Personality Scale (DS-14) which includes a measure of NA, the Brief COPE which measures coping styles, and the Perceived Stress Scale.
Scores on the AAQ-II had moderate positive relationships with the MEAQ total score and all MEAQ subscales with the exception of distress endurance which had a moderate negative relationship. The AAQ-II had a stronger relationship with NA, avoidant coping, and perceived stress than did the MEAQ. All MEAQ subscales had a positive relationship to NA, avoidant coping, and perceived stress with the exception of distress endurance which had a negative relationship with these constructs. While the AAQ-II is limited as a unitary measure of EA the multiple dimensions of the MEAQ may involve an extraneous factor of distress endurance. Future work should examine the relationships of the MEAQ with NA, avoidant coping and perceived stress with clinical populations.
Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of ...gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8⁺ T cells and show that it is distinct from functional memory CD8⁺ T cells. Exhausted CD8⁺ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing snows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8⁺ T cells.
Decline of peak viremia during acute HIV-1 infection occurs before the development of vigorous adaptive immunity, and the level of decline correlates inversely with the rate of AIDS progression, ...implicating a potential role for the innate immune response in determining disease outcome. The combined expression of an activating natural killer (NK) cell receptor, the killer immunoglobulin-like receptor (KIR) 3DS1, and its presumed ligand, human leukocyte antigen (HLA)-B Bw4-80I, has been associated in epidemiological studies with a slow progression to AIDS. We examined the functional ability of NK cells to differentially control HIV-1 replication in vitro based on their KIR and HLA types. NK cells expressing KIR3DS1 showed strong, significant dose- and cell contact-dependent inhibition of HIV-1 replication in target cells expressing HLA-B Bw4-80I compared with NK cells that did not express KIR3DS1. Furthermore, KIR3DS1+ NK cells and NKLs were preferentially activated, and lysed HIV-1 infected target cells in an HLA-B Bw4-80I-dependent manner. These data provide the first functional evidence that variation at the KIR locus influences the effectiveness of NK cell activity in the containment of viral replication.
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