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.

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.

Acute Administration of HIV-1 Tat Protein Drives Glutamatergic Alterations in a Rodent Model of HIV-Associated Neurocognitive Disorders.

HIV-1-associated neurocognitive disorders (HAND) are a major comorbidity of HIV-1 infection, marked by impairment of executive function varying in severity. HAND affects nearly half of people living with HIV (PLWH), with mild forms predominating since the use of anti-retroviral therapies (ART). The HIV-1 transactivator of transcription (Tat) protein is found in the cerebrospinal fluid of patients adherent to ART, and its administration or expression in animals causes cognitive symptoms. Studies of Tat interaction with the N-methyl-D-aspartate receptor (NMDAR) suggest that glutamate toxicity contributes to Tat-induced impairments. To identify changes in regional glutamatergic circuitry underlying cognitive impairment, we injected recombinant Tat86 or saline to medial prefrontal cortex (mPFC) of male Sprague-Dawley rats. Rats were assessed with behavioral tasks that involve intact functioning of mPFC including the novel object recognition (NOR), spatial object recognition (SOR), and temporal order (TO) tasks at 1 and 2 postoperative weeks. Following testing, mPFC tissue was collected and analyzed by RT-PCR. Results showed Tat86 in mPFC-induced impairment in SOR, and upregulation of Grin1 and Grin2a transcripts. To further understand the mechanism of Tat toxicity, we assessed the effects of full-length Tat101 on gene expression in mPFC by RNA sequencing. The results of RNAseq suggest that glutamatergic effects of Tat86 are maintained with Tat101, as Grin2a was upregulated in Tat101-injected tissue, among other differentially expressed genes. Spatial learning and memory impairment and Grin2a upregulation suggest that exposure to Tat protein drives adaptation in mPFC, altering the function of circuitry supporting spatial learning and memory.

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.

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.

Roles of neuropathology-associated reactive astrocytes: a systematic review.

In the contexts of aging, injury, or neuroinflammation, activated microglia signaling with TNF-α, IL-1α, and C1q induces a neurotoxic astrocytic phenotype, classified as A1, A1-like, or neuroinflammatory reactive astrocytes. In contrast to typical astrocytes, which promote neuronal survival, support synapses, and maintain blood-brain barrier integrity, these reactive astrocytes downregulate supportive functions and begin to secrete neurotoxic factors, complement components like C3, and chemokines like CXCL10, which may facilitate recruitment of immune cells across the BBB into the CNS. The proportion of pro-inflammatory reactive astrocytes increases with age through associated microglia activation, and these pro-inflammatory reactive astrocytes are particularly abundant in neurodegenerative disorders. As the identification of astrocyte phenotypes progress, their molecular and cellular effects are characterized in a growing array of neuropathologies.

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.

[Essen transition model for neuromuscular diseases]. / "Essener Transitionsmodell" bei neuromuskulären Erkrankungen.

BACKGROUND: Advances in healthcare systems with new therapeutic options improve the life expectancy of patients with neuromuscular diseases. With this, a shift in the phenotype of the diseases from the neuromuscular system towards other organs is more frequently observed, requiring closer interdisciplinary cooperation in caring for these young adults. Therefore, the transition to the adult caring system is nowadays a multilayered transfer with the need for complex care of these patients. OBJECTIVE: How can the transitional process be efficiently structured to combine the therapeutic effort of each specialist discipline involved and improve the healthcare process and quality of life in young adults with neuromuscular diseases? MATERIAL AND METHOD: The Departments of Neuropediatrics and Neurology of the University Medicine Essen established the Essen transition model for a structured transitional process. A concept of care was developed for the late onset Pompe's disease, Duchenne muscular dystrophy and juvenile myasthenia gravis representatively for neuromuscular diseases. It consists of four components: 1) In a standardized operational procedure (SOP), general processes, clinical diagnostic steps and guidance of treatment between the two departments are harmonized and specified. 2) The young adults and their relatives are seen in a joint consultation of both disciplines allowing a comprehensive handover for healthcare professionals. 3) In a quarterly meeting, transition conference representatives from the different specialized disciplines from pediatric and adult medicine get together for a case-related interdisciplinary exchange. 4) An interdepartmental transitional database was created to integrate all diagnostic results and parameters as a common information platform and data basis. CONCLUSION: The Essen transition model aims to close a gap in the transition of patients with neuromuscular diseases and improve healthcare in these patients with their complex needs.

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.

Essen transition model for neuromuscular diseases.

BACKGROUND: With the optimization of medical care structures and the rapid progress in the development of new therapeutic methods, an increase in life expectancy is observed in patients with neuromuscular diseases. This leads to an expansion of the phenotypic spectrum, whereby new or previously less relevant disease manifestations in different organ systems gain more importance. The care of adolescents and young adults with neuromuscular diseases, therefore, requires increasingly close interdisciplinary collaboration within neuromuscular centers. RESEARCH QUESTION: How can the transition process from pediatric to adult care be structured so that the individual disciplines are efficiently integrated into the complex treatment and care process, and the patients' quality of life is improved? MATERIAL AND METHODS: A structured transition process was established at the University Hospital in Essen, Germany. Exemplarily, a comparable care concept was developed based on Pompe disease, Duchenne muscular dystrophy, and juvenile myasthenia gravis comprising four elements: (1) With the introduction of cross-department standard operating procedures, the logistical processes, as well as the diagnostic and therapeutic measures, are uniformly coordinated, and the transition process is bindingly defined. (2) To ensure a seamless transition, young patients are seen with their parents during joint consultations before they reach their 17th birthday. This creates an opportunity for patients to get to know the subsequent department structure and build a lasting relationship of trust. (3) A quarterly "transition board" regularly brings together the participating disciplines from pediatric and adult care systems for a case-related interdisciplinary exchange and continuous optimization of the transition process. (4) A cross-department "Transition Database", in which medical findings and parameters are recorded, was implemented as a common information platform and database. CONCLUSION: The Essen Transition Model aims to close the gap in care for young patients with neuromuscular diseases during the critical transition from pediatric to adult medicine and to create a successful continuation of treatment in adulthood.

Chronic Low Dose Morphine Does Not Alter Two In Vitro BBB Models.

The blood-brain barrier (BBB) mediates cellular and molecular passage between the central nervous system (CNS) and peripheral circulation. Compromised BBB integrity has been linked to neurocognitive deficits in multiple diseases and various infections, including those associated with HIV-1 infection. Understanding the impact of exposure to pharmaceuticals, such as those utilized for pain management by patients suffering from CNS disease, on BBB regulation and function is clinically important. In this study, we modelled two different BBB systems; a primary human co-culture and a cell line monoculture. These systems were both exposed to three daily repeat doses of morphine and examined for alterations to BBB integrity via permeability, PBMC transmigration, and chemokine gradient changes. We did not find any significant changes to either BBB system with repeat morphine dosing, suggesting that repeat morphine exposure may not play a significant role in BBB changes.

The impossibility of mortality evaluation of skin cancer screening in Germany based on health insurance data: a case-control study.

BACKGROUND: The aim of this paper was to perform a mortality evaluation of skin cancer screening (SCS) in Germany using General Local Health Insurance Fund (AOK) data. METHODS: AOK-insured men and women aged 35-99 years who died of cutaneous malignant melanoma in 2015-2016 were identified. Controls were AOK-insured people who survived to the end of each case's year of death. For each case, 10 controls were matched. The SCS history of each individual was reconstructed using the billing codes 01745 and 01746. RESULTS: In total, 1037 melanoma deaths and 10,370 controls were included. Cumulative SCS prevalence increased among controls over calendar years, as expected (males and females, 2009: 13.5% and 12.5%; 2015: 52.1% and 55.1%). In contrast, among cases, cumulative SCS prevalence was already high in 2009 and did not show a monotonic increase over the years of diagnosis. Of the 1037 melanoma deaths, 224 (21.6%) had at least one SCS settled in the 12 months after diagnosis. DISCUSSION: A mortality evaluation with health insurance data alone is not possible because SCS billing codes are not only used for real SCS but also for occasion-related diagnostic work-up of abnormal skin findings. A mortality evaluation with health insurance data requires an individual linking with data of the screening physician and the cancer registries.

One-year follow-up healthcare costs of patients diagnosed with skin cancer in Germany: a claims data analysis.

BACKGROUND: Routine skin cancer screening (SCS) is covered by the German statutory health insurance (SHI) since 2008. The objective of this study was to compare direct healthcare costs between patients in whom skin cancer was detected by routine SCS and patients in whom skin cancer was not detected by routine SCS. METHODS: A retrospective observational study of administrative claims data from a large German SHI was performed. Patients with a diagnosis of malignant melanoma (MM) or non-melanoma skin cancer (NMSC) diagnosed in 2014 or 2015 were included. Costs were obtained for one year before and one year after diagnosis and analyzed in a difference-in-differences approach using regression models. Frequency matching was applied and risk adjustment was performed. Additional analyses were conducted, separately for specific age groups, excluding persons who died during the observation period and without taking costs for screening into consideration. RESULTS: A total of 131,801 patients were included, of whom 13,633 (10.3%) had a diagnosis of MM and 118,168 (89.7%) had a diagnosis of NMSC. The description of total costs (without risk adjustment) shows lower mean total costs among patients whose skin cancer was detected via routine SCS compared to patients in whom skin cancer was not detected by routine SCS (MM: €5,326 (95% confidence interval (CI) €5,073; €5,579) vs. €9,038 (95% CI €8,629; €9,448); NMSC: €4,660 (95% CI €4,573; €4,745) vs. €5,890 (95% CI €5,813; €5,967)). Results of the regression analysis show cost savings of 18.8% (95% CI -23.1; -8.4) through routine SCS for patients with a diagnosis of MM. These cost savings in MM patients were more pronounced in patients younger than 65 years of age. For patients with a diagnosis of NMSC, the analysis yields a non-substantial increase in costs (2.5% (95% CI -0.1; 5.2)). CONCLUSION: Cost savings were detected for persons with an MM diagnosed by routine SCS. However, the study could not detect lower costs due to routine SCS in the large fraction of persons with a diagnosis of NMSC. These results offer important insights into the cost structure of the routine SCS and provide opportunities for refinements.

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.

Heterogeneity of national legislation and practice on clinical trials with vulnerable patients based on the EU Clinical Trials Directive by the example of adults permanently incapable of giving informed consent.

In principle, persons wishing to participate in a clinical trial must give informed consent in advance after comprehensive information has been provided. Under certain conditions, it is possible to deviate from this requirement in the European Union (EU) in order to enable the participation of so-called vulnerable persons who are incapable of giving their informed consent. Kuthning et al. [1] have already dealt with general and specific aspects of vulnerable patients and the principle of informed consent in clinical trials. One group of vulnerable persons, for example, are adults temporarily or permanently incapable of giving consent due to their state of health. For a long period of time, no systematic and uniform legal basis for clinical trials existed in the EU as a whole. The Clinical Trials Directive (CTD) [2], adopted in 2001, aimed to change this by harmonizing all legal regulations on clinical trials applicable in the EU, but nevertheless allowing national deviations in implementation into national laws through opening clauses and aspects that were left unregulated. In view of the Clinical Trials Regulation (CTR) [3] which, according to the current status, will with high probability be applied from 2022 on, and which in future will be the legal basis for clinical trials with medicinal products in humans, applied directly in all EU member states, the necessity to take stock of the effects of the CTD was evident. The national deviations with regard to the participation of patients incapable of giving informed consent were investigated qualitatively and quantitatively by means of a systematic analysis of legislation in 16 EU countries and a retrospective database analysis of a European clinical trial registry over a ten-year observation period. Although the analysis initially showed a predominantly homogeneous picture, the differences between the EU member states became apparent in a detailed examination. The database analysis yielded a clear result, since in some countries the majority of clinical trials are carried out. The clearest difference was found between the legal analysis and the results of the evaluated clinical trials concerning adults who are permanently incapable of giving informed consent. A presumed association between the "degree of liberality" of the national law and the frequency of clinical trials conducted in the respective country could not be confirmed. In the past, the selection of countries for conducting a clinical trial was based less on legal requirements and more on experience and financial considerations.

Nusinersen treatment in adult patients with spinal muscular atrophy: a safety analysis of laboratory parameters.

BACKGROUND: Nusinersen is an intrathecally administered antisense oligonucleotide (ASO) that improves motor function in patients with spinal muscular atrophy (SMA). In addition to efficacy, the safety of a therapy is the decisive factor for the success of the treatment. For some ASOs, various organ toxicities have been described, such as thrombocytopenia, renal and liver impairment, or coagulation abnormalities. However, systematic data on laboratory parameters under treatment with nusinersen are mainly available from studies in infants and children. Therefore, our aim was to assess the safety of nusinersen therapy in adult SMA patients. METHODS: Laboratory data from 404 nusinersen injections performed in 50 adult patients with SMA type 2 and type 3 were retrospectively analyzed. RESULTS: The total observation period was 76.9 patient-years, and patients received up to 12 injections. Our data provides no new safety concerns. In cerebrospinal fluid (CSF), the mean white blood cell count and lactate remained stable over time. Total CSF protein increased by 2.9 mg/dL. No change in mean platelet count was observed under therapy. Only one patient showed sporadic mild thrombocytopenia. Coagulation parameters and inflammatory markers were stable. The mean creatinine level decreased by 0.09 mg/dL. Analysis of mean liver enzyme levels revealed no relevant changes during treatment. CONCLUSION: Our data demonstrate a favorable safety profile of nusinersen therapy in adult SMA patients under longer-term "real-world" conditions. In particular, we found no evidence of clinically relevant platelet declines, coagulopathies, or renal or hepatic organ toxicities, which are common concerns with the use of ASOs.

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.

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.

Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy.

Globally, human immunodeficiency virus type 1 (HIV-1) infection is a major health burden for which successful therapeutic options are still being investigated. Challenges facing current drugs that are part of the established life-long antiretroviral therapy (ART) include toxicity, development of drug resistant HIV-1 strains, the cost of treatment, and the inability to eradicate the provirus from infected cells. For these reasons, novel anti-HIV-1 therapeutics that can prevent or eliminate disease progression including the onset of the acquired immunodeficiency syndrome (AIDS) are needed. While development of HIV-1 vaccination has also been challenging, recent advancements demonstrate that infection of HIV-1-susceptible cells can be prevented in individuals living with HIV-1, by targeting C-C chemokine receptor type 5 (CCR5). CCR5 serves many functions in the human immune response and is a co-receptor utilized by HIV-1 for entry into immune cells. Therapeutics targeting CCR5 generally involve gene editing techniques including CRISPR, CCR5 blockade using antibodies or antagonists, or combinations of both. Here we review the efficacy of these approaches and discuss the potential of their use in the clinic as novel ART-independent therapies for HIV-1 infection.

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.