Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.

Despite increasing data demonstrating dopamine as an inflammatory mediator of the innate immune system, the molecular mechanisms underlying its effects in human cells remain incompletely defined. Here, we define an unrecognized pathway in which dopamine induces robust IL-6 secretion in primary human monocyte-derived macrophages (hMDMs) through mitochondrial stress. Dopamine initiates a transient mitochondrial membrane depolarization that leads to sustained alterations in mitochondrial dynamics, including morphology and metabolism, in a time-dependent manner. These events promote the mtDNA release into the cytoplasm, triggering cGAS-STING pathway and downstream NF-κB signaling. Pharmacological inhibition at multiple nodes of this pathway attenuates IL-6 secretion, establishing mitochondrial dysfunction and cGAS-STING signaling as central mediators of dopamine-driven IL6 secretion. Variability in dopamine receptor expression across donors correlates with the magnitude of IL-6 responses. Together, these findings redefine the interface between dopamine signaling and systemic inflammation and highlight an unrecognized source of inter-individual variation in immune responses.

Latent human immunodeficiency virus type 1 (HIV-1) reservoirs in the central nervous system (CNS) may sustain viral persistence and neuroinflammation contributing to HIV-associated neurocognitive disorders (HAND) despite suppressive ART. AAV9-delivered CRISPR has successfully edited SIV proviral DNA in peripheral tissues with acceptable safety profiles, but the extent of in vivo genome editing in the brain remains unclear. Using SIV-infected rhesus macaques, we mapped intact proviral DNA across CNS regions and tested systemic AAV9-CRISPR/Cas9 targeting conserved sites within Ψ packaging signal and Gag region. Ten adult rhesus macaques were infected with genetically barcoded SIVmac239, suppressed with ART, then randomized to receive intravenous AAV9-SaCas9 with dual gRNAs (Ψ+Gag) or a Cas9-only control. At necropsy after viral rebound, SIV genomes were detected in multiple brain regions as well as lymphoid tissues, confirming the CNS as a persistent reservoir during ART. Barcode analysis revealed region-specific patterns consistent with compartmentalized CNS persistence. In CRISPR-treated animals, proviral editing was measurable across anatomically distinct CNS sites. These findings demonstrate that intact and potentially replication-competent virus persists in the primate brain under ART and that systemic AAV9-CRISPR can reach and edit proviral DNA in this sanctuary, supporting genome editing as a strategy toward durable remission of CNS reservoirs.

Classic toll-like receptor activation on macrophages drives inflammation, and our data indicate that the neurotransmitter dopamine can also drive inflammation in these cells. Our data in human monocyte derived macrophages (MDM) show that dopamine promotes an inflammatory phenotype. We also show in vitro culture environment can impact MDM function. We hypothesized that dopamine receptor (DR) expression patterns impact MDM inflammatory response, and that serum supplementation in vitro can alter this. We cultured MDM in DMEM or RPMI Medium +/- FBS or Macrophage Serum Free Media (M-SFM). MDM were untreated or stimulated with dopamine or LPS and assessed for changes in DR expression (RT-qPCR), cytokine production (AlphaLISA), transcriptional profile (RNA-seq), NF-kB activity, and morphology (High Content Imaging). Dopamine increased IL-6 secretion which correlated with the expression pattern of specific DR transcripts. Serum influenced MDM activation, with FBS supplementation and culture in M-SFM significantly altered the baseline transcriptome. M-SFM culture also significantly suppressed the NF-kB response to LPS while the impact of FBS supplementation on pharmacodynamics was related to the base media. Culture media and serum supplementation also drove morphological differences (area, length-to-width, perimeter-to-area ratio). These studies show dopamine drives inflammation in MDM and that in vitro microenvironment is an active component of experimentation. NIH DA057337 (PJG), NIH DA058051 (PJG), T32-MH079785 (supporting BC), F30AI179472 (BC), P30-MH092177 (WD), and the Department of Pharmacology and Physiology at Drexel University College of Medicine. Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

In the context of neurodegeneration, activated microglia facilitate inflammation via secretion of TNF-α, IL-1α, and C1q. Astrocytes exposed to this signaling array polarize to a reactive inflammatory phenotype, termed A1 or A1-like. Astrocytes are essential for neuronal survival, synaptic support, and blood–brain barrier (BBB) function, but A1-like astrocytes upregulate inflammatory gene expression, downregulate neurotrophic factors, and secrete neurotoxic signals. The consequences of A1-like polarization on BBB function are unknown but may have etiological implications for some diseases. Frequently identified by upregulation of complement component 3 (C3), A1-like astrocytes have been characterized in neurodegenerative disorders like Alzheimer’s disease, with polarization correlated with disease progression and severity. However, the role of A1-like astrocytes in neurodegeneration associated with chronic viral infections, like HIV-1-associated neurocognitive disorder (HAND), remains unclear. An in vitro system using primary human astrocytes, as well as a BBB model featuring primary human brain microvascular endothelial cells (BMECs) co-cultured with astrocytes, was used to elucidate cellular and molecular consequences of chronic astrocyte activation. As measured by whole transcriptome analysis and protein expression assays, repeated treatment with TNF-α, IL-1α, and C1q induced A1-like polarization of astrocytes both in monoculture and in a BBB model, resulting in increased secretion of pro-inflammatory signals. No substantial change to BBB permeability was observed. In contrast, exposure to HIV-1 viral protein Tat did not independently induce A1-like polarization. Ongoing investigations into the effect of astrocyte polarization on BBB integrity and treatment with pathogenic proteins may provide insights into the role of neurotoxic astrocytes in neurovirologic pathologies.

Non-thermal plasma, cold plasma, and atmospheric-pressure plasma are few terms used to describe the plasma used in plasma medicine research. The resulting ambiguity hampers literature searches, confuses discussion, and complicates collaborations. To assess the full breadth of this problem, we designed a natural language processing model (NLP) that surveyed approximately 15,000 papers in response to the query “plasma medicine” indexed in PubMed between 2020-2022. Our NLP was constructed and executed using the Hugging Face transformers API and PubMed BERT pretrained model. We used this model to determine the prevalence and to assess the utility of each term for searching literature relevant to plasma medicine. The effectiveness of each term was measured by precision, the ability to discriminate relevant and irrelevant literature; and recall, the ability to retrieve relevant literature. Each term was given a combined effectiveness score of 0-1 (1 = ideal effectiveness) accounting for precision, recall, sample size, and model confidence. Our model showed that of the twelve commonly used terms analyzed, none received a combined effectiveness score over 0.025. We concluded that there is no universal term for “plasma” that provides a satisfactory representation of literature. These results highlight the need for standardization of nomenclature in plasma medicine.

Macrophages are central to innate immunity and are routinely used in vitro to examine molecular mechanisms contributing to innate immune signaling. However, there is a lack of consensus within the field for optimal in vitro culturing methods, and it is not well understood whether differences in culture conditions produce incongruent outcomes. Here, we compared the effects of commonly used culture medium compositions on TLR4-mediated proinflammatory activity in primary human monocyte-derived macrophages (hMDMs) isolated from healthy blood donors. hMDMs were cultured in fetal bovine serum (FBS)-containing or FBS-free conditions in either Dulbecco's Modified Eagle Medium (DMEM), RPMI, or in Macrophage-Serum Free Medium (M-SFM). Lipopolysaccharide-mediated immune response was measured through nuclear factor κB activation and cytokine and chemokine secretion, which were muted in M-SFM cultures compared with DMEM and RPMI cultures. FBS supplementation increased total cytokine secretion in response to lipopolysaccharide but also showed higher baseline secretion, suggesting a proinflammatory phenotype. Moreover, M-SFM cultures exhibited less phagocytosis compared with DMEM and RPMI cultures. Morphologic analysis of unstimulated hMDMs revealed the highest cell area and length-to-width ratio in M-SFM compared with DMEM or RPMI cultures. FBS-free and M-SFM conditions produced distinct transcriptional profiles compared with media supplemented with FBS, most notably in cell cycle pathways and lipid homeostasis, respectively. Overall, DMEM and RPMI produce comparable morphologic and functional results, albeit with some small differences, while M-SFM produces a muted inflammatory response in macrophages. These data demonstrate that in vitro microenvironment drives differential inflammatory outcomes in human macrophages and is a critical component of experimental design in this cell type.

Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability.Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential.Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV.Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.

Purpose of review The leading gene editing strategy for a human immunodeficiency virus type 1 (HIV-1) cure involves the delivery of SaCas9 and two guide RNAs (gRNAs) in an adeno-associated viral (AAV) vector. As a dual-component system, CRISPR is targeted to a genetic locus through the choice of a Cas effector and gRNA protospacer design pair. As CRISPR research has expanded in recent years, these components have been investigated for utilization in cure strategies, which will be discussed in this article. Recent findings Type II SpCas9 and SaCas9 have been the leading Cas effectors across gene editing therapeutics to date. Additionally, extensive research has expanded the potential to multiplex gRNAs and target them effectively to the highly genetically diverse HIV-1 provirus. More recently, the Type V family of Cas12 effectors opens a new opportunity to use a smaller Cas protein for packaging into an AAV vector with multiplexed gRNAs. Summary In understanding the individual components of a CRISPR/Cas therapeutic cure for HIV-1, it is important to know that the currently used strategies can be improved upon. Future areas will include alternative smaller Cas effectors, multiplexed gRNAs designs, and/or alternative delivery modalities.

Human immunodeficiency virus type 1 (HIV-1) infection is well known as one of the most complex and difficult viral infections to cure. The difficulty in developing curative strategies arises in large part from the development of latent viral reservoirs (LVRs) within anatomical and cellular compartments of a host. The clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system shows remarkable potential for the inactivation and/or elimination of integrated proviral DNA within host cells, however, delivery of the CRISPR/Cas9 system to infected cells is still a challenge. In this review, the main factors impacting delivery, the challenges for delivery to each of the LVRs, and the current successes for delivery to each reservoir will be discussed.

Human immunodeficiency virus-associated neurocognitive disorders persist in the combination antiretroviral therapy era. CD4 nadir is a well-established predictor of cognition cross-sectionally, but its impact on longitudinal neurocognitive (NC) trajectories is unclear. The few studies on this topic examined trajectories of global cognition, rather than specific NC domains. The current study examined CD4 nadir in relation to domain-specific NC decline. 132 HIV + adults from the Temple/Drexel Comprehensive NeuroHIV Center, Clinical and Translational Research Support Core Cohort were administered comprehensive NC assessments longitudinally, with last visit occurring an average of 12 years after CD4 nadir. Linear mixed models were used to examine CD4 nadir in relation to longitudinal NC trajectories in three empirically identified NC domains: speed/executive function (S/EF), visuospatial memory (VM), and verbal fluency (VF). CD4 nadir was associated with change in VF (p = 0.020), but not with S/EF or VM. Specifically, those with CD4 nadir < 200 demonstrated increasing VF over time (p = .002), whereas those with CD4 nadir > 200 demonstrated stable VF (p = .568), though these differing trajectories may partly reflect regression to the mean or differential practice effect. CD4 dynamics over time were analyzed as potential mechanisms for the identified associations, with mixed findings. While low CD4 nadir has been associated with weaker neurocognition among people living with HIV, the results of this study suggest that low CD4 nadir is not associated with ongoing decline a decade later. Nadir-related deficits in VF may be stable or even improve over time, possibly reflecting the beneficial cognitive effects of long-term treatment and immune reconstitution.

Throughout the human immunodeficiency virus (HIV) epidemic, neuropathogenic properties of the virus have become an important aspect of the disease process. In the early years, this was thought to be in large part due to the central nervous system (CNS) viral load. As combination antiretroviral therapy (ART) became more effective and viral load in the CNS was decreased, the severity of neurocognitive impairment decreased but it did not decrease the percentage of individuals with this neurologic comorbidity. Infection in the CNS occurs in the acute stage of infection with the establishment of a CNS viral reservoir. Viruses within this reservoir are thought to evolve in a compartmentalized manner, making the variants and potential pathogenesis of the viruses in the CNS unique to that compartment. Currently, neurocognitive impairment is thought to be the result of smoldering chronic viral infection where the viral load is kept low or undetectable with viral proteins produced over long periods of time resulting in disruption of intracellular dysfunction and deregulation of intercellular communication pathways. This cellular dysfunction is both in the infected cell populations and within the uninfected cell populations as intra- versus extracellular proteins, respectively. Infected cells include the perivascular macrophage, microglia, and astrocyte and cells of the blood–brain barrier, whereas the uninfected cells include those four cell populations as well as neurons. The combination of effects between these cells results in dysregulation of cytokines, chemokines, ROS, glutamate balance, etc. The HIV proteins shown to individually and likely in combination cause neurotoxicity are Tat, Vpr, Nef, and gp120. With the exception of gp120, these are all viral regulatory proteins. As such, this chapter will focus on the role of these viral proteins in neuropathogenic process following viral CNS invasion.

In people living with HIV-1 (PLWH), antiretroviral therapy (ART) eventually becomes necessary to suppress the emergence of human immunodeficiency virus type 1 (HIV-1) replication from latent reservoirs because HIV-1-specific immune responses in PLWH are suboptimal. Immunotherapies that enhance anti-HIV-1 immune responses for better control of virus reemergence from latent reservoirs are postulated to offer ART-free control of HIV-1. Toward the goal of developing an HIV-1-specific immunotherapy based on non-thermal plasma (NTP), the early immunological responses to NTP-exposed latently infected T lymphocytes were examined. Application of NTP to the J-Lat T-lymphocyte cell line (clones 10.6 and 15.4) stimulated monocyte recruitment and macrophage maturation, which are key steps in initiation of an immune response. In contrast, CD8+ T lymphocytes in a mixed lymphocyte reaction assay were not stimulated by the presence of NTP-exposed J-Lat cells. Furthermore, co-culture of NTP-exposed J-Lat cells with mature phagocytes did not modulate their antigen presentation to primary CD8+ T lymphocytes (cross-presentation). However, reactivation from latency was stimulated in a clone-specific manner by NTP. Overall, these studies, which demonstrated that ex vivo application of NTP to latently infected lymphocytes can stimulate key immune cell responses, advance the development of an NTP-based immunotherapy that will provide ART-free control of HIV-1 reactivation in PLWH.

Objective:Human immunodeficiency virus (HIV) type 1 (HIV-1), cardiovascular disease, and HIV-associated neurocognitive disorders (HAND) disproportionately affect Black/African American individuals compared to other racial and ethnic groups. Understanding the mechanisms of cognitive health disparities is essential for developing policy and health interventions to combat such disparities. Cardiovascular risk factors/diseases are common comorbidities that likely contribute to cognitive health disparities among Black/African American people living with HIV (PWH), but their impacts on cognition longitudinally in this population are unclear. The current study examines the relationship between cardiovascular risk and cognitive functioning over time in Black/African American adults living with HIV.Participants and Methods:A sample of 122 Black/African American adults with HIV (ages 25-68, M=51.8, SD=7.7; 98% on antiretroviral therapy; 91% with undetectable viral load) were selected from the Drexel/Temple Comprehensive NeuroHIV Center, Clinical and Translational Research Support Core (CTRSC; based at Drexel University College of Medicine) Cohort. They completed longitudinal visits (300 total visits, average follow-up time=4.9 years) that included clinical interviews, medical record review, biometric measurements, and comprehensive neuropsychological assessments. Cardiovascular risk factors of interest were body mass index (BMI), waist-to-height ratio (WHtR), and a total vascular risk burden score (VBS) representing five risk factors: obesity, central obesity, diabetes, hyperlipidemia, and hypertension. Based on a prior principal component analysis, three cognitive domains were examined: (1) verbal fluency, (2) visual memory/visuoconstruction, and (3) motor speed/executive functions. Mixed models were used to examine domain-specific cognitive trajectories in relation to baseline cardiovascular risk factors and changes in cardiovascular risk factors.Results:Overall, cognitive test performance improved over time (pConclusions:Higher total vascular risk burden was associated with less favorable verbal fluency trajectories, reflecting the negative cognitive consequences of disorders such as diabetes, hyperlipidemia, and hypertension. Unexpectedly, greater increases in BMI and WHtR were associated with more favorable trajectories in motor speed and executive functioning. In this population, weight gain may be a proxy for other positive health factors, such as immune reconstitution, which will be examined in future analyses. Taken together, cardiovascular risk factors have heterogeneous associations with cognitive trajectories, emphasizing the importance of examining the mechanisms of these varying relationships. Future research will examine how social determinants of health, such as racial/ethnic discrimination, contribute to disparities in cardiovascular risk factors and cognitive outcomes.

Verbal memory was related to better MMT-R performance, but neither cognition nor MMT-R were good predictors of detectable vs. undetectable viral load. However, among those with low-level viral replication, poorer visuospatial memory and difficulty with simple MMT-R items were preliminarily related to higher viral load. Given the small sample, results are limited by low statistical power. Future research will further explore relationships among cognition, MMT-R, RNA, and regimen complexity in larger samples to inform development of tools to monitor adherence difficulties.