CD4 nadir and neurocognitive trajectories in people living with HIV.

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.

Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency.

Defining the variables that impact the specificity of CRISPR/Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targeted loci has also been hypothesized to be an important factor. In this study, functional data from two genome-wide assays, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors that modulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency.

Specific amino acids in HIV-1 Vpr are significantly associated with differences in patient neurocognitive status.

Even in the era of combination antiretroviral therapies used to combat human immunodeficiency virus type 1 (HIV-1) infection, up to 50 % of well-suppressed HIV-1-infected patients are still diagnosed with mild neurological deficits referred to as HIV-associated neurocognitive disorders (HAND). The multifactorial nature of HAND likely involves the HIV-1 accessory protein viral protein R (Vpr) as an agent of neuropathogenesis. To investigate the effect of naturally occurring variations in Vpr on HAND in well-suppressed HIV-1-infected patients, bioinformatic analyses were used to correlate peripheral blood-derived Vpr sequences with patient neurocognitive performance, as measured by comprehensive neuropsychological assessment and the resulting Global Deficit Score (GDS). Our studies revealed unique associations between GDS and the presence of specific amino acid changes in peripheral blood-derived Vpr sequences [neuropsychological impairment Vpr (niVpr) variants]. Amino acids N41 and A55 in the Vpr sequence were associated with more pronounced neurocognitive deficits (higher GDS). In contrast, amino acids I37 and S41 were connected to measurably lower GDS. All niVpr variants were also detected in DNA isolated from HIV-1-infected brain tissues. The implication of these results is that niVpr variants alter the genesis and/or progression of HAND through differences in Vpr-mediated effects in the peripheral blood and/or the brain.

Utilization of HIV-1 envelope V3 to identify X4- and R5-specific Tat and LTR sequence signatures.

BACKGROUND: HIV-1 entry is a receptor-mediated process directed by the interaction of the viral envelope with the host cell CD4 molecule and one of two co-receptors, CCR5 or CXCR4. The amino acid sequence of the third variable (V3) loop of the HIV-1 envelope is highly predictive of co-receptor utilization preference during entry, and machine learning predictive algorithms have been developed to characterize sequences as CCR5-utilizing (R5) or CXCR4-utilizing (X4). It was hypothesized that while the V3 loop is predominantly responsible for determining co-receptor binding, additional components of the HIV-1 genome may contribute to overall viral tropism and display sequence signatures associated with co-receptor utilization. RESULTS: The accessory protein Tat and the HlV-1 long terminal repeat (LTR) were analyzed with respect to genetic diversity and compared by Jensen-Shannon divergence which resulted in a correlation with both mean genetic diversity as well as the absolute difference in genetic diversity between R5- and X4-genome specific trends. As expected, the V3 domain of the gp120 protein was enriched with statistically divergent positions. Statistically divergent positions were also identified in Tat amino acid sequences within the transactivation and TAR-binding domains, and in nucleotide positions throughout the LTR. We further analyzed LTR sequences for putative transcription factor binding sites using the JASPAR transcription factor binding profile database and found several putative differences in transcription factor binding sites between R5 and X4 HIV-1 genomes, specifically identifying the C/EBP sites I and II, and Sp site III to differ with respect to sequence configuration for R5 and X4 LTRs. CONCLUSION: These observations support the hypothesis that co-receptor utilization coincides with specific genetic signatures in HIV-1 Tat and the LTR, likely due to differing transcriptional regulatory mechanisms and selective pressures applied within specific cellular targets during the course of productive HIV-1 infection.

Prolonged Morphine Exposure Induces Increased Firm Adhesion in an in Vitro Model of the Blood-Brain Barrier.

The blood-brain barrier (BBB) has been defined as a critically important protective barrier that is involved in providing essential biologic, physiologic, and immunologic separation between the central nervous system (CNS) and the periphery. Insults to the BBB can cause overall barrier damage or deregulation of the careful homeostasis maintained between the periphery and the CNS. These insults can, therefore, yield numerous phenotypes including increased overall permeability, interendothelial gap formation, alterations in cytokine and chemokine secretion, and accelerated cellular passage. The current studies expose the human brain microvascular endothelial cell line, hCMEC/D3, to prolonged morphine exposure and aim to uncover the mechanisms underlying alterations in barrier function in vitro. These studies show alterations in the mRNA and protein levels of the cellular adhesion molecules (CAMs) intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and activated leukocyte cell adhesion molecule that correlate with an increased firm adhesion of the CD3⁺ subpopulation of peripheral blood mononuclear cells (PBMCs). Overall, these studies suggest that prolonged morphine exposure may result in increased cell migration into the CNS, which may accelerate pathological processes in many diseases that involve the BBB.

What's in a cure: designing a broad-spectrum HIV gene therapy.

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.

Delivering CRISPR to the HIV-1 reservoirs.

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.

Attenuation of Forkhead signaling by the retinal determination factor DACH1.

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant ellipse, encodes a key component of the retinal determination gene network that governs cell fate. Herein, cyclic amplification and selection of targets identified a DACH1 DNA-binding sequence that resembles the FOX (Forkhead box-containing protein) binding site. Genome-wide in silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP coupled with high-throughput sequencing mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the cyclic amplification and selection of targets-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis.

Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy.

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.

Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.

Immunomodulatory Effects of Non-Thermal Plasma in a Model for Latent HIV-1 Infection: Implications for an HIV-1-Specific Immunotherapy.

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.

Examining virtual driving test performance and its relationship to individuals with HIV-associated neurocognitive disorders.

Significance: Existing screening tools for HIV-associated neurocognitive disorders (HAND) are often clinically impractical for detecting milder forms of impairment. The formal diagnosis of HAND requires an assessment of both cognition and impairment in activities of daily living (ADL). To address the critical need for identifying patients who may have disability associated with HAND, we implemented a low-cost screening tool, the Virtual Driving Test (VDT) platform, in a vulnerable cohort of people with HIV (PWH). The VDT presents an opportunity to cost-effectively screen for milder forms of impairment while providing practical guidance for a cognitively demanding ADL. Objectives: We aimed to: (1) evaluate whether VDT performance variables were associated with a HAND diagnosis and if so; (2) systematically identify a manageable subset of variables for use in a future screening model for HAND. As a secondary objective, we examined the relative associations of identified variables with impairment within the individual domains used to diagnose HAND. Methods: In a cross-sectional design, 62 PWH were recruited from an established HIV cohort and completed a comprehensive neuropsychological assessment (CNPA), followed by a self-directed VDT. Dichotomized diagnoses of HAND-specific impairment and impairment within each of the seven CNPA domains were ascertained. A systematic variable selection process was used to reduce the large amount of VDT data generated, to a smaller subset of VDT variables, estimated to be associated with HAND. In addition, we examined associations between the identified variables and impairment within each of the CNPA domains. Results: More than half of the participants (N = 35) had a confirmed presence of HAND. A subset of twenty VDT performance variables was isolated and then ranked by the strength of its estimated associations with HAND. In addition, several variables within the final subset had statistically significant associations with impairment in motor function, executive function, and attention and working memory, consistent with previous research. Conclusion: We identified a subset of VDT performance variables that are associated with HAND and assess relevant functional abilities among individuals with HAND. Additional research is required to develop and validate a predictive HAND screening model incorporating this subset.

Large-scale integration of microarray data reveals genes and pathways common to multiple cancer types.

The global gene expression analysis of cancer and healthy tissues typically results in large numbers of genes that are significantly altered in cancer. Such data, however, has been difficult to interpret due to the high level of variation of gene lists across laboratories and the small sample sizes used in individual studies. In this investigation, we compiled microarray data obtained from the same platform family from 84 laboratories, resulting in a database containing 1,043 healthy tissue samples and 4,900 cancer samples for 13 different tissue types. The primary cancers considered included adrenal gland, brain, breast, cervix, colon, kidney, liver, lung, ovary, pancreas, prostate and skin tissues. We normalized the data together and analyzed subsets for the discovery of genes involved in normal to cancer transformation. Our integrated significance analysis of microarrays approach produced top 400 gene lists for each of the 13 cancer types. These lists were highly statistically enriched with genes already associated with cancer in research publications excluding microarray studies (p < 1.31 E - 12). The genes MTIM and RRM2 appeared in nine and TOP2A in eight lists of significantly altered genes in cancer. In total, there were 132 genes present in at least four gene lists, 11 of which were not previously associated with cancer. The list contains 17 metal ions and 15 adenyl ribonucleotide binding proteins, six kinases and six transcription factors. Our results point to the value of integrating microarray data in the study of combination drug therapies targeting metastasis.

Commentary to Gusenbauer and Haddaway 2020: Evaluating retrieval qualities of Google Scholar and PubMed.

We read with considerable interest the study by Gusenbauer and Haddaway (Gusenbauer and Haddaway, 2020, Research Synthesis Methods, doi:10.1002/jrsm.1378) comparing the systematic search qualities of 28 search systems, including Google Scholar (GS) and PubMed. Google Scholar and PubMed are the two most popular free academic search tools in biology and chemistry, with GS being the number one search tool in the world. Those academics using GS as their principal system for literature searches may be unaware of research which enumerates five critical features for scientific literature tools that greatly influenced Gusenbauer's 2020 study. Using this list as the framework for a targeted comparison between just GS and PubMed, we found stark differences which overwhelmingly favored PubMed. In this comment, we show that by comparing the characteristics of the two search tools, features that are particularly useful in one search tool, but are missing in the other, are strikingly spotlighted. One especially popular feature that ubiquitously appears in GS, but not in PubMed, is the forward citation search found under every citation as a clickable Cited by N link. We seek to improve the PubMed search experience using two approaches. First, we request that PubMed add Cited by N links, making them as omnipresent as the GS links. Second, we created an open-source command-line tool, pmidcite, which is used alongside PubMed to give information to researchers to help with the choice of the next paper to examine, analogous to how GS's Cited by N links help to guide users. Find pmidcite at https://github.com/dvklopfenstein/pmidcite.

HIV-1 cure strategies: why CRISPR?

INTRODUCTION: Antiretroviral therapy (ART) has transformed prognoses for HIV-1-infected individuals but requires lifelong adherence to prevent viral resurgence. Targeted elimination or permanent deactivation of the latently infected reservoir harboring integrated proviral DNA, which drives viral rebound, is a major focus of HIV-1 research. AREAS COVERED: This review covers the current approaches to developing curative strategies for HIV-1 that target the latent reservoir. Discussed herein are shock and kill, broadly neutralizing antibodies (bNAbs), block and lock, Chimeric antigen receptor (CAR) T cells, immune checkpoint modulation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) coreceptor ablation, and CRISPR/Cas9 proviral excision. Emphasis is placed on CRISPR/Cas9 proviral excision/inactivation. Recent advances and future directions toward discovery and translation of HIV-1 therapeutics are discussed. EXPERT OPINION: CRISPR/Cas9 proviral targeting fills a niche amongst HIV-1 cure strategies by directly targeting the integrated provirus without the necessity of an innate or adaptive immune response. Each strategy discussed in this review has shown promising results with the potential to yield curative or adjuvant therapies. CRISPR/Cas9 is singular among these in that it addresses the root of the problem, integrated proviral DNA, with the capacity to permanently remove or deactivate the source of HIV-1 recrudescence.

Off-Target Analysis in Gene Editing and Applications for Clinical Translation of CRISPR/Cas9 in HIV-1 Therapy.

As genome-editing nucleases move toward broader clinical applications, the need to define the limits of their specificity and efficiency increases. A variety of approaches for nuclease cleavage detection have been developed, allowing a full-genome survey of the targeting landscape and the detection of a variety of repair outcomes for nuclease-induced double-strand breaks. Each approach has advantages and disadvantages relating to the means of target-site capture, target enrichment mechanism, cellular environment, false discovery, and validation of bona fide off-target cleavage sites in cells. This review examines the strengths, limitations, and origins of the different classes of off-target cleavage detection systems including anchored primer enrichment (GUIDE-seq), in situ detection (BLISS), in vitro selection libraries (CIRCLE-seq), chromatin immunoprecipitation (ChIP) (DISCOVER-Seq), translocation sequencing (LAM PCR HTGTS), and in vitro genomic DNA digestion (Digenome-seq and SITE-Seq). Emphasis is placed on the specific modifications that give rise to the enhanced performance of contemporary techniques over their predecessors and the comparative performance of techniques for different applications. The clinical relevance of these techniques is discussed in the context of assessing the safety of novel CRISPR/Cas9 HIV-1 curative strategies. With the recent success of HIV-1 and SIV-1 viral suppression in humanized mice and non-human primates, respectively, using CRISPR/Cas9, rigorous exploration of potential off-target effects is of critical importance. Such analyses would benefit from the application of the techniques discussed in this review.

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.

Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection.

Effective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH.

Designing Safer CRISPR/Cas9 Therapeutics for HIV: Defining Factors That Regulate and Technologies Used to Detect Off-Target Editing.

Human immunodeficiency virus type-1 (HIV-1) infection has resulted in the death of upward of 39 million people since being discovered in the early 1980s. A cure strategy for HIV-1 has eluded scientists, but gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offer a new approach to developing a cure for HIV infection. While the CRISPR/Cas9 system has been used successfully in a number of different types of studies, there remains a concern for off-target effects. This review details the different aspects of the Cas9 system and how they play a role in off-target events. In addition, this review describes the current technologies available for detecting off-target cleavage events and their advantages and disadvantages. While some studies have utilized whole genome sequencing (WGS), this method sacrifices depth of coverage for interrogating the whole genome. A number of different approaches have now been developed to take advantage of next generation sequencing (NGS) without sacrificing depth of coverage. This review highlights four widely used methods for detecting off-target events: (1) genome-wide unbiased identification of double-stranded break events enabled by sequencing (GUIDE-Seq), (2) discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-Seq), (3) circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-Seq), and (4) breaks labeling in situ and sequencing (BLISS). Each of these technologies has advantages and disadvantages, but all center around capturing double-stranded break (DSB) events catalyzed by the Cas9 endonuclease. Being able to define off-target events is crucial for a gene therapy cure strategy for HIV-1.

Integrated Human Immunodeficiency Virus Type 1 Sequence in J-Lat 10.6.

The full length of HIV/R7/E-/GFP integrated in the J-Lat 10.6 cell line was sequenced in this study. The single copy of the integrated virus, including the breakpoints from the human chromosome to the provirus, was amplified by two separate PCRs. A 10,200-bp genome sequence was acquired, analyzed, and deposited in GenBank.