Lee Ann Campbell

The CRISPR/Cas9 system is a revolutionary gene editing technology that combines simplicity of use and efficiency of mutagenesis. As this technology progresses toward human therapies, valid concerns including off-target mutations and immunogenicity must be addressed. One approach to address these issues is to minimize the presence of the CRISPR/Cas9 components by maintaining a tighter temporal control of Cas9 endonuclease and reducing the time period of activity. This has been achieved to some degree by delivering the CRISPR/Cas9 system via pre-formed Cas9 + gRNA ribonucleoprotein (RNP) complexes. In this review, we first discuss the molecular modifications that can be made using CRISPR/Cas9 and provide an overview of current methods for delivering Cas9 RNP complexes both in vitro and in vivo. We conclude with examples of how Cas9 RNP delivery may be used to target neuroinflammatory processes, namely in regard to viral infections of the central nervous system and neurodegenerative diseases. We propose that Cas9 RNP delivery is a viable approach when considering the CRISPR/Cas9 system for both experimentation and the treatment of disease. Graphical Abstract

Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.

The chemokineCCL5 prevents neuronal cell death mediated both by amyloid β, as well as the human immunodeficiency virus viral proteins gp120 and Tat. BecauseCCL5 binds toCCR5,CCR3 and/orCCR1 receptors, it remains unclear which of these receptors plays a role in neuroprotection. Indeed,CCL5 also has neuroprotective activity in cells lacking these receptors.CCL5 may bind to a G‐protein‐coupled receptor 75 (GPR75), which encodes for a 540 amino‐acid orphan receptor of the Gqα family. In this study, we have usedSH‐SY5Y human neuroblastoma cells to characterize whetherCCL5 could activate a Gq signaling throughGPR75. BothqPCRand flow cytometry show that these cells expressGPR75 but do not expressCCR5,CCR3 orCCR1 receptors.SY‐SY5Y cells were then used to examineCCL5‐mediated signaling. We report thatCCL5 promotes a time‐ and concentration‐dependent phosphorylation of protein kinase B (AKT), glycogen synthase kinase 3β, and extracellular signal–regulated kinase (ERK) 1/2. Specific antagonists ofCCR5,CCR3, andCCR1 did not preventCCL5 from increasing phosphorylatedAKTorERK. Moreover,CCL5 promotes a time‐dependent internalization ofGPR75. Lastly, knocking downGPR75 expression by aCRISPR‐Cas9 approach inhibited the ability ofCCL5 to activatepERKinSH‐SY5Y cells. Therefore, we propose thatGPR75 is a novel receptor forCCL5 that could explain some of the pharmacological action of this chemokine. These findings may help in the development of small moleculeGPR75 agonists that mimicCCL5.Open Practices: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found athttps://cos.io/our-services/open-science-badges/. image

Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of HIV‐associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro‐inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV provirus capable of recreating several hallmarks of HIV infection. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology and integrated a modified HIV provirus into CHME‐5 immortalized microglia to create HIV‐NanoLuc CHME‐5. In the modified provirus, the Gag‐Pol region is replaced with the coding region for NanoLuciferase (NanoLuc), which allows for the rapid assay of HIV long terminal repeat activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV‐NanoLuc CHME‐5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro‐inflammatory factors lipopolysaccharide (LPS) and tumor necrosis factor alpha and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the toll‐like receptor‐4 gene by CRISPR/Cas9 reduced LPS‐mediated proviral activation, and pharmacological application of NF‐κB inhibitor sulfasalazine similarly diminished proviral activity. Overall, these data suggest that HIV‐NanoLuc CHME‐5 may be a useful tool in the study of HIV‐mediated neuropathology and proviral regulation.

Morphine has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in the brain. However, little is known about the effect of morphine withdrawal on BDNF and its precursor protein, or proBDNF, which induces neuronal apoptosis. In this work, we examined whether BDNF and proBDNF levels change in rats chronically injected with escalating doses of morphine and those who undergo spontaneous withdrawal for 60 h. We observed, in the frontal cortex and striatum, that the ratio of BDNF to proBDNF changed depending upon the experimental paradigm. Morphine treatment and morphine withdrawal increased both BDNF and proBDNF levels. However, the increase in proBDNF immunoreactivity in withdrawal rats was more robust than that observed in morphine-treated rats. proBDNF is processed either intracellularly by furin or extracellularly by the tissue plasminogen activator (tPA)/plasminogen system or matrix metalloproteases (MMPs). To examine the mechanisms whereby chronic morphine treatment and morphine withdrawal differentially affects BDNF/proBDNF, the levels MMP-3 and MMP-7, furin, and tPA were analyzed. We found that morphine increases tPA levels, whereas withdrawal causes a decrease. To confirm the involvement of tPA in the morphine-mediated effect on BDNF/proBDNF, we exposed cortical neurons to morphine in the presence of the tPA inhibitor plasminogen activator inhibitor-1 (PAI-1). This inhibitor reversed the morphine-mediated decrease in proBDNF, supporting the hypothesis that morphine increases the availability of BDNF by promoting the extracellular processing of proBDNF by tPA. Because proBDNF could negatively influence synaptic repair, preventing withdrawal is crucial for reducing neurotoxic mechanisms associated with opioid abuse.