Recent advances in virology, particularly in the study of HIV-1, have significantly progressed the pursuit of a definitive cure for the disease. Emerging therapeutic strategies encompass innovative gene-editing technologies, immune-modulatory interventions, and next-generation antiretroviral agents. Efforts to eliminate or control viral reservoirs have also gained momentum, with the aim of achieving durable viral remission without the continuous requirement for antiretroviral therapy. Despite these promising developments, critical challenges persist in bridging the gap between laboratory findings and clinical implementation. This review provides a comprehensive analysis of recent breakthroughs, ongoing clinical trials, and the barriers that must be addressed to translate these advancements into effective treatments, emphasizing the multifaceted approaches being pursued to achieve a curative solution for HIV-1 infection.

The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection.

Although combination antiretroviral therapy (ART) has been a landmark achievement for the treatment of human immunodeficiency virus (HIV), an HIV cure has remained elusive. Elimination of latent HIV reservoirs that persist throughout HIV infection is the most challenging barrier to an HIV cure. The progressive HIV infection is marked by the increasing size and diversity of latent HIV reservoirs until an effective immune response is mobilized, which can control but not eliminate HIV infection. The stalemate between HIV replication and the immune response is manifested by the establishment of a viral set point. ART initiation during the early stage limits HIV reservoir development, preserves immune function, improves the quality of life, and may lead to ART-free viral remission in a few people living with HIV (PLWH). However, for the overwhelming majority of PLWH, early ART initiation alone does not cure HIV, and lifelong ART is needed to sustain viral suppression. A critical area of research is focused on determining whether HIV could be functionally cured if additional treatments are provided alongside early ART. Several HIV interventions including Block and Lock, Shock and Kill, broadly neutralizing antibody (bNAb) therapy, adoptive CD8+ T cell therapy, and gene therapy have demonstrated delayed viral rebound and/or viral remission in animal models and/or some PLWH. Whether or not their application during early infection can improve the success of HIV remission is less studied. Herein, we review the current state of clinical and investigative HIV interventions and discuss their potential to improve the likelihood of post-treatment remission if initiated during early infection.

The efforts to discover HIV therapeutics have continued since the first human immunodeficiency virus (HIV) infected patient was confirmed in the 1980s. Ten years later, the first HIV drug, zidovudine (AZT), targeting HIV reverse transcriptase, was developed. Meanwhile, scientists were enlightened to discover new drugs that target different HIV genes, like integrase, protease, and host receptors. Combination antiretroviral therapy (cART) is the most feasible medical intervention to suppress the virus in people with HIV (PWH) and control the epidemic. ART treatment has made HIV a chronic infection rather than a fatal disease, but ART does not eliminate latent reservoirs of HIV-1 from the host cells; strict and life-long adherence to ART is required for the therapy to be effective in patients. In this review, we first discussed the scientific history of conventional HIV drug discovery since scientists need to develop more and more drugs to solve drug-resistant issues and release the side effects. Then, we summarized the novel research technologies, like gene editing, applied to HIV treatment and their contributions to eliminating HIV as a complementary therapy.

Durable HIV-1 remission has been reported in a person who received allogeneic stem cell transplants (SCTs) involving CCR5 Δ32/Δ32 donor cells. Much of the reduction in HIV-1 burden following allogeneic SCT with or without donor cells inherently resistant to HIV-1 infection is likely due to cytotoxic graft-versus-host effects on residual recipient immune cells. Nonetheless, there has been growing momentum to develop and implement stem cell therapies that lead to durable long-term antiretroviral therapy (ART)-free remission without the need for SCT.

Most current research leverages gene editing techniques to modify hematopoietic stem cells which differentiate into immune cells capable of harboring HIV-1. Approaches include targeting genes that encode HIV-1 co-receptors using Zinc Finger Nucleases (ZFN) or CRISPR-Cas-9 to render a pool of adult or progenitor cells resistant to de-novo infection. Other strategies involve harnessing multipotent mesenchymal stromal cells to foster immune environments that can more efficiently recognize and target HIV-1 while promoting tissue homeostasis.

Many of these strategies are currently in a state of infancy or adolescence; nonetheless, promising preclinical and first-in-human studies have been performed, providing further rationale to focus resources on stem cell therapies.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated enzyme-CAS holds great promise for treating many uncured human diseases and illnesses by precisely correcting harmful point mutations and disrupting disease-causing genes. The recent Food and Drug Association (FDA) approval of the first CRISPR-based gene therapy for sickle cell anemia marks the beginning of a new era in gene editing. However, delivering CRISPR specifically into diseased cells in vivo is a significant challenge and an area of intense research. The identification of new CRISPR/Cas variants, particularly ultra-compact CAS systems with robust gene editing activities, paves the way for the low-capacity delivery vectors to be used in gene therapies. CRISPR/Cas technology has evolved beyond editing DNA to cover a wide spectrum of functionalities, including RNA targeting, disease diagnosis, transcriptional/epigenetic regulation, chromatin imaging, high-throughput screening, and new disease modeling. CRISPR/Cas can be used to engineer B-cells to produce potent antibodies for more effective vaccines and enhance CAR T-cells for the more precise and efficient targeting of tumor cells. However, CRISPR/Cas technology has challenges, including off-target effects, toxicity, immune responses, and inadequate tissue-specific delivery. Overcoming these challenges necessitates the development of a more effective and specific CRISPR/Cas delivery system. This entails strategically utilizing specific gRNAs in conjunction with robust CRISPR/Cas variants to mitigate off-target effects. This review seeks to delve into the intricacies of the CRISPR/Cas mechanism, explore progress in gene therapies, evaluate gene delivery systems, highlight limitations, outline necessary precautions, and scrutinize the ethical considerations associated with its application.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

During the past 20 years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector-mediated gene addition, inborn errors of immunity are excellent candidate diseases for a corrective autologous hematopoietic stem cell gene editing strategy. Research on gene editing for inborn errors of immunity is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of inborn errors of immunity. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.

The clustered regularly interspaced short palindromic repeats (CRISPR) system, a rapidly advancing genome editing technology, allows DNA alterations into the genome of organisms. Gene editing using the CRISPR system enables more precise and diverse editing, such as single nucleotide conversion, precise knock-in of target sequences or genes, chromosomal rearrangement, or gene disruption by simple cutting. Moreover, CRISPR systems comprising transcriptional activators/repressors can be used for epigenetic regulation without DNA damage. Stem cell DNA engineering based on gene editing tools has enormous potential to provide clues regarding the pathogenesis of diseases and to study the mechanisms and treatments of incurable diseases. Here, we review the latest trends in stem cell research using various CRISPR/Cas technologies and discuss their future prospects in treating various diseases.

The CRISPR/Cas system, an innovative gene-editing tool, is emerging as a promising technique for genome modifications. This straightforward technique was created based on the prokaryotic adaptive immune defense mechanism and employed in the studies on human diseases that proved enormous therapeutic potential. A genetically unique patient mutation in the process of gene therapy can be corrected by the CRISPR method to treat diseases that traditional methods were unable to cure. However, introduction of CRISPR/Cas9 into the clinic will be challenging because we still need to improve the technology's effectiveness, precision, and applications. In this review, we first describe the function and applications of the CRISPR-Cas9 system. We next delineate how this technology could be utilized for gene therapy of various human disorders, including cancer and infectious diseases and highlight the promising examples in the field. Finally, we document current challenges and the potential solutions to overcome these obstacles for the effective use of CRISPR-Cas9 in clinical practice.

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.

Acute Lymphoblastic Leukemia (ALL) is the predominant hematological malignancy in pediatric populations, originating from B- or T-cell precursors within the bone marrow. The disease exhibits a high degree of heterogeneity, both at the molecular level and in terms of clinical presentation. A complex interplay between inherited and acquired genetic alterations contributes to disease pathogenesis, often resulting in the disruption of cellular functions integral to the leukemogenic process. The advent of CRISPR/Cas9 as a gene editing tool has revolutionized biological research, underscoring its potential to modify specific genomic loci implicated in cancer. Enhanced understanding of molecular alterations in ALL has facilitated significant advancements in therapeutic strategies. In this review, we scrutinize the application of CRISPR/Cas9 as a tool for identifying genetic targets to improve therapy, circumvent drug resistance, and facilitate CAR-T cell-based immunotherapy. Additionally, we discuss the challenges and future prospects of CRISPR/Cas9 applications in ALL.

Human immunodeficiency virus (HIV) is known to cause hematological malignancy. Hematopoietic stem cell transplantation (HPSCT) is an advanced treatment for that. Currently, there are three successful HIV-eliminated cases, and two received HPSCT from CCR5-absent donors. It is well established that the CCR5 protein on the cell surface assists human immunodeficiency virus entry. Preliminary studies have revealed that knocking out CCR5 and/or CXCR4 may inhibit the viral entry of HIV, which may prove promising in the further development of HIV treatment options. Herein, we suggest performing autologous or allogeneic HSCT with CCR5 KO hematopoietic stem cells in patients who suffer from complicated HIV conditions, particularly drug-resistant HIV or a concurrent diagnosis of HIV with lymphoma/leukemia, to achieve complete HIV remission. Nevertheless, at the clinical forefront of CRISPR-HIV technology, more efforts should be directed to advance nonhuman primate (NHP) models for studies of HIV pathogenesis and off-target assessments within this system. CRISPR-Cas9 knock out of host HSCT-expressing CCR5 or CXCR4 may confer HIV-resistance, which when applied to bedside therapeutics in an allogeneic or autologous manner can warrant a permanent and effective treatment outcome.

The CRISPR/Cas9 system is a powerful genome editing tool that has made enormous impacts on next-generation molecular diagnostics and therapeutics, especially for genetic disorders that traditional therapies cannot cure. Currently, CRISPR-based gene editing is widely applied in basic, preclinical, and clinical studies. In this review, we attempt to identify trends in clinical studies involving CRISPR techniques to gain insights into the improvement and contribution of CRISPR/Cas technologies compared to traditional modified modalities. The review of clinical trials is focused on the applications of the CRISPR/Cas systems in the treatment of cancer, hematological, endocrine, and immune system diseases, as well as in diagnostics. The scientific basis underlined is analyzed. In addition, the challenges of CRISPR application in disease therapies and recent advances that expand and improve CRISPR applications in precision medicine are discussed.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is focused on the current CRISPR/Cas9 landscape, e.g., on Cas9 variants with improved properties, on Cas9-derived and fusion proteins, on Cas9 delivery methods, on pre-existing immunity against CRISPR/Cas9 proteins, anti-CRISPR proteins, and their possible roles in CRISPR/Cas9 function improvement. Moreover, this review presents a detailed outline of CRISPR/Cas9-based diagnostics and therapeutic approaches. Finally, the review addresses the future expansion of genome editors' toolbox with Cas9 orthologs and other CRISPR/Cas proteins.

Comparative genomics has enabled the discovery of tiny clustered regularly interspaced short palindromic repeat (CRISPR) bacterial immune system effectors with enormous potential for manipulating eukaryotic genomes. Recently, smaller Cas proteins, including miniature Cas9, Cas12, and Cas13 proteins, have been identified and validated as efficient genome editing and base editing tools in human cells. The compact size of these novel CRISPR effectors is highly desirable for generating CRISPR-based therapeutic approaches, mainly to overcome in vivo delivery constraints, providing a promising opportunity for editing pathogenic mutations of clinical relevance and knocking down RNAs in human cells without inducing chromosomal insertions or genome alterations. Thus, these tiny CRISPR-Cas systems represent new and highly programmable, specific, and efficient platforms, which expand the CRISPR toolkit for potential therapeutic opportunities.

The evolution of drug-resistant viral strains and anatomical and cellular reservoirs of HIV pose significant clinical challenges to antiretroviral therapy. CCR5 is a coreceptor critical for HIV host cell fusion, and a homozygous 32-bp gene deletion (∆32) leads to its loss of function. Interestingly, an allogeneic HSCT from an HIV-negative ∆32 donor to an HIV-1-infected recipient demonstrated a curative approach by rendering the recipient's blood cells resistant to viral entry. Ex vivo gene editing tools, such as CRISPR/Cas9, hold tremendous promise in generating allogeneic HSC grafts that can potentially replace allogeneic ∆32 HSCTs. Here, we review antiretroviral therapeutic challenges, clinical successes, and failures of allogeneic and allogeneic ∆32 HSCTs, and newer exciting developments within CCR5 editing using CRISPR/Cas9 in the search to cure HIV.

The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.

Translational medicine aims to improve human health by exploring potential treatment methods developed during basic scientific research and applying them to the treatment of patients in clinical settings. The advanced perceptions of gene functions have remarkably revolutionized clinical treatment strategies for target agents. However, the progress in gene editing therapy has been hindered due to the severe off-target effects and limited editing sites. Fortunately, the development in the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system has renewed hope for gene therapy field. The CRISPR-Cas9 system can fulfill various simple or complex purposes, including gene knockout, knock-in, activation, interference, base editing, and sequence detection. Accordingly, the CRISPR-Cas9 system is adaptable to translational medicine, which calls for the alteration of genomic sequences. This review aims to present the latest CRISPR-Cas9 technology achievements and prospect to translational medicine advances. The principle and characterization of the CRISPR-Cas9 system are firstly introduced. The authors then focus on recent pre-clinical and clinical research directions, including the construction of disease models, disease-related gene screening and regulation, and disease treatment and diagnosis for multiple refractory diseases. Finally, some clinical challenges including off-target effects, in vivo vectors, and ethical problems, and future perspective are also discussed.

Chimeric antigen receptor (CAR) T cells and natural killer (NK) cells are genetically engineered immune cells that can detect target antigens on the surface of target cells and eliminate them following adoptive transfer. Recent progress in CAR-based therapies has led to outstanding clinical success in certain patients with leukemias and lymphomas and offered therapeutic benefits to those resistant to conventional therapies. The universal approach to stable CAR transgene delivery into the T/NK cells is the use of viral particles. Such approaches mediate semi-random transgene insertions spanning the entire genome with a high preference for integration into sites surrounding highly-expressed genes and active loci. Regardless of the variable CAR expression level based on the integration site of the CAR transgene, foreign integrated DNA fragments may affect the neighboring endogenous genes and chromatin structure and potentially change a transduced T/NK cell behavior and function or even favor cellular transformation. In contrast, site-specific integration of CAR constructs using recent genome-editing technologies could overcome the limitations and disadvantages of universal random gene integration. Herein, we explain random and site-specific integration of CAR transgenes in CAR-T/NK cell therapies. Also, we tend to summarize the methods for site-specific integration as well as the clinical outcomes of certain gene disruptions or enhancements due to CAR transgene integration. Also, the advantages and limitations of using site-specific integration methods are discussed in this review. Ultimately, we will introduce the genomic safe harbor (GSH) standards and suggest some appropriate safety prospects for CAR integration in CAR-T/NK cell therapies.

Hematopoietic stem cells (HSCs) are known for their significant capability to reconstitute and preserve a functional hematopoietic system in long-term periods after transplantation into conditioned hosts. HSCs are thus crucial cellular targets for the continual repair of inherited hematologic, metabolic, and immunologic disorders. In addition, HSCs can undergo various fates, such as apoptosis, quiescence, migration, differentiation, and self-renewal. Viruses continuously pose a remarkable health risk and request an appropriate, balanced reaction from our immune system, which as well as affects the bone marrow (BM). Therefore, disruption of the hematopoietic system due to viral infection is essential. In addition, patients for whom the risk-to-benefit ratio of HSC transplantation (HSCT) is acceptable have seen an increase in the use of HSCT in recent years. Hematopoietic suppression, BM failure, and HSC exhaustion are all linked to chronic viral infections. Virus infections continue to be a leading cause of morbidity and mortality in HSCT recipients, despite recent advancements in the field. Furthermore, whereas COVID-19 manifests initially as an infection of the respiratory tract, it is now understood to be a systemic illness that significantly impacts the hematological system. Patients with advanced COVID-19 often have thrombocytopenia and blood hypercoagulability. In the era of COVID-19, Hematological manifestations of COVID-19 (i.e., thrombocytopenia and lymphopenia), the immune response, and HSCT may all be affected by the SARS-CoV-2 virus in various ways. Therefore, it is important to determine whether exposure to viral infections may affect HSCs used for HSCT, as this, in turn, may affect engraftment efficiency. In this article, we reviewed the features of HSCs, and the effects of viral infections on HSCs and HSCT, such as SARS-CoV-2, HIV, cytomegalovirus, Epstein-Barr virus, HIV, etc. Video Abstract.

In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.

The increasing incidence of sexually transmitted diseases in women, including human papillomavirus (HPV) infection, has led to the need to develop user-friendly potential prevention methods. At present, although there are several therapeutic parts, none of them has a preventive effect, but they are only limited to providing patients with symptom relief. Researchers have now recognized the need to find effective local preventive agents. One of the potential undiscovered local fungicides is the vaginal delivery of CRISPR/Cas9. CRISPR/Cas9 delivery involves silencing gene expression in a sequence-specific manner in the pathogenic agent, thus showing microbicidal activity. However, vaginal mucosal barrier and physiological changes (such as pH value and variable epithelial thickness in the menstrual cycle) are the main obstacles to effective delivery and cell uptake of CRISPR/Cas9. To enhance the vaginal delivery of CRISPR/Cas9, so far, nano-carrier systems such as lipid delivery systems, macromolecular systems, polymer nanoparticles, aptamers, and cell-penetrating peptides have been extensively studied. In this paper, various nano-carriers and their prospects in the preclinical stage are described, as well as the future significance of CRISPR/Cas9 vaginal delivery based on nano-carriers.

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure.

Lessons learned from decades-long practice in the transplantation of hematopoietic stem and progenitor cells (HSPCs) to treat severe inherited disorders or cancer, have set the stage for the current ex vivo gene therapies using autologous gene-modified hematopoietic stem and progenitor cells that have treated so far, hundreds of patients with monogenic disorders. With increased knowledge of hematopoietic stem and progenitor cell biology, improved modalities for patient conditioning and with the emergence of new gene editing technologies, a new era of hematopoietic stem and progenitor cell-based gene therapies is poised to emerge. Gene editing has the potential to restore physiological expression of a mutated gene, or to insert a functional gene in a precise locus with reduced off-target activity and toxicity. Advances in patient conditioning has reduced treatment toxicities and may improve the engraftment of gene-modified cells and specific progeny. Thanks to these improvements, new potential treatments of various blood- or immune disorders as well as other inherited diseases will continue to emerge. In the present review, the most recent advances in hematopoietic stem and progenitor cell gene editing will be reported, with a focus on how this approach could be a promising solution to treat non-blood-related inherited disorders and the mechanisms behind the therapeutic actions discussed.

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.

In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice. Due to their advantageous features, the CRISPR-Cas systems are already employed in several biological and medical research fields as the most suitable technique for genome engineering. In this review, we aim to describe the CRISPR-Cas systems that have been identified among prokaryotic organisms and engineered for genome manipulation studies. Furthermore, a comprehensive comparison between the innovative CRISPR-Cas methodology and the previously utilized ZFN and TALEN editing nucleases is also discussed. Ultimately, we highlight the contribution of CRISPR-Cas methodology in modern biomedicine and the current plethora of available applications for gene KO, repression and/or overexpression, as well as their potential implementation in therapeutical strategies that aim to improve patients' quality of life.

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.

In recent years, gene-editing technologies have revolutionised precision medicine, and human trials of this technology have been reported in cell-based cancer therapies and other genetic disorders. The same techniques have the potential to reverse mutations in monogenic primary immunodeficiencies (PIDs), and transplantation of edited haematopoietic stem cells may provide a functional cure for these diseases. In this review, we discuss the methods of gene editing being explored and describe progress made so far with several PIDs. We also detail the remaining challenges, how to confidently detect off-target effects and chromosomal abnormalities in a timely manner, how to obtain long-term benefits, and how to achieve physiological levels of expression of the therapeutic gene. With advances in gene editing, we envisage a robust clinical translation of this technology in the coming decade.

CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34+ hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination.

CRISPR-Cas is a versatile genome editing technology that has been broadly applied in both basic research and translation medicine. Ever since its discovery, the bacterial derived endonucleases have been engineered to a collection of robust genome-editing tools for introducing frameshift mutations or base conversions at site-specific loci. Since the initiation of first-in-human trial in 2016, CRISPR-Cas has been tested in 57 cell therapy trials, 38 of which focusing on engineered CAR-T cells and TCR-T cells for cancer malignancies, 15 trials of engineered hematopoietic stem cells treating hemoglobinopathies, leukemia and AIDS, and 4 trials of engineered iPSCs for diabetes and cancer. Here, we aim to review the recent breakthroughs of CRISPR technology and highlight their applications in cell therapy.

Treatment of genetic disorders by genomic manipulation has been the unreachable goal of researchers for many decades. Although our understanding of the genetic basis of genetic diseases has advanced tremendously in the last few decades, the tools developed for genomic editing were not efficient and practical for their use in the clinical setting until now. The recent advancements in the research of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems offered an easy and efficient way to edit the genome and accelerated the research on their potential use in the treatment of genetic disorders. In this review, we summarize the clinical trials that evaluate the CRISPR/Cas systems for treating different genetic diseases and highlight promising preclinical research on CRISPR/Cas mediated treatment of a great diversity of genetic disorders. Ultimately, we discuss the future of CRISPR/Cas mediated genome editing in genetic diseases.

Modern-day hematopoietic stem cell (HSC) therapies, such as gene therapy, modify autologous HSCs prior to re-infusion into myelo-conditioned patients and hold great promise for treatment of hematological disorders. While this approach has been successful in numerous clinical trials, it relies on transplantation of ex vivo modified patient HSCs, which presents several limitations. It is a costly and time-consuming procedure, which includes only few patients so far, and ex vivo culturing negatively impacts on the viability and stem cell-properties of HSCs. If viral vectors are used, this carries the additional risk of insertional mutagenesis. A therapy delivered to HSCs in vivo, with minimal disturbance of the HSC niche, could offer great opportunities for novel treatments that aim to reverse disease symptoms for hematopoietic disorders and could bring safe, effective and affordable genetic therapies to all parts of the world. However, substantial unmet needs exist with respect to the in vivo delivery of therapeutics to HSCs. In the last decade, in particular with the development of gene editing technologies such as CRISPR/Cas9, nanoparticles (NPs) have become an emerging platform to facilitate the manipulation of cells and organs. By employing surface modification strategies, different types of NPs can be designed to target specific tissues and cell types in vivo. HSCs are particularly difficult to target due to the lack of unique cell surface markers that can be utilized for cell-specific delivery of therapeutics, and their shielded localization in the bone marrow (BM). Recent advances in NP technology and genetic engineering have resulted in the development of advanced nanocarriers that can deliver therapeutics and imaging agents to hematopoietic stem- and progenitor cells (HSPCs) in the BM niche. In this review we provide a comprehensive overview of NP-based approaches targeting HSPCs to control and monitor HSPC activity in vitro and in vivo, and we discuss the potential of NPs for the treatment of malignant and non-malignant hematological disorders, with a specific focus on the delivery of gene editing tools.

Although the world is currently focused on the COVID-19 pandemic, HIV/AIDS remains a significant threat to public health. To date, the HIV/AIDS pandemic has claimed the lives of over 36 million people, while nearly 38 million people are currently living with the virus. Despite the undeniable success of antiretroviral therapy (ART) in controlling HIV, the medications are not curative. Soon after initial infection, HIV integrates into the genome of infected cells as a provirus, primarily, within CD4+ T lymphocytes and tissue macrophages. When not actively transcribed, the provirus is referred to as a latent reservoir because it is hidden to the immune system and ART. Following ART discontinuation, HIV may emerge from the replication-competent proviruses and resumes the infection of healthy cells. Thus, these latent reservoirs are a major obstacle to an HIV cure, and their removal remains a priority. A vital aspect in the development of curative therapies is the demonstration of efficacy in an animal model, such as the humanized mouse model. Therefore, optimization, standardization, and validation of the humanized mouse model are a priority. The purpose of this review article is to provide an update on existing humanized mouse models, highlighting the advantages and disadvantages of each as they pertain to HIV cure studies and to review the approaches to curative therapies that are under investigation.

Epigenetic dysregulation is an important determinant of many pathological conditions and diseases. Designer molecules that can specifically target endogenous DNA sequences provide a means to therapeutically modulate gene function. The prokaryote-derived CRISPR/Cas editing systems have transformed our ability to manipulate the expression program of genes through specific DNA and RNA targeting in living cells and tissues. The simplicity, utility, and robustness of this technology have revolutionized epigenome editing for research and translational medicine. Initial success has inspired efforts to discover new systems for targeting and manipulating nucleic acids on the epigenetic level. The evolution of nuclease-inactive and RNA-targeting Cas proteins fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions opened up an unprecedented level of possibilities for the development of "next-generation" gene therapy therapeutics. The rational design and construction of different types of designer molecules paired with viral-mediated gene-to-cell transfers, specifically using lentiviral vectors (LVs) and adeno-associated vectors (AAVs) are reviewed in this paper. Furthermore, we explore and discuss the potential of these molecules as therapeutic modulators of endogenous gene function, focusing on modulation by stable gene modification and by regulation of gene transcription. Notwithstanding the speedy progress of CRISPR/Cas-based gene therapy products, multiple challenges outlined by undesirable off-target effects, oncogenicity and other virus-induced toxicities could derail the successful translation of these new modalities. Here, we review how CRISPR/Cas-based gene therapy is translated from research-grade technological system to therapeutic modality, paying particular attention to the therapeutic flow from engineering sophisticated genome and epigenome-editing transgenes to delivery vehicles throughout efficient and safe manufacturing and administration of the gene therapy regimens. In addition, the potential solutions to some of the obstacles facing successful CRISPR/Cas utility in the clinical research are discussed in this review. We believe, that circumventing these challenges will be essential for advancing CRISPR/Cas-based tools towards clinical use in gene and cell therapies.

The development of gene-editing technology has been one of the biggest advances in biomedicine over the past two decades. Not only can it be used as a research tool to build a variety of disease models for the exploration of disease pathogenesis at the genetic level, it can also be used for prevention and treatment. This is done by intervening with the expression of target genes and carrying out precise molecular targeted therapy for diseases. The simple and flexible clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technology overcomes the limitations of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). For this reason, it has rapidly become a preferred method for gene editing. As a new gene intervention method, CRISPR/Cas9 has been widely used in the clinical treatment of tumours and rare diseases; however, its application in the field of cardiovascular diseases is currently limited. This article reviews the application of the CRISPR/Cas9 editing technology in cardiovascular disease research and treatment, and discusses the limitations and prospects of this technology.

The expanding genome editing toolbox has revolutionized life science research ranging from the bench to the bedside. These "molecular scissors" have offered us unprecedented abilities to manipulate nucleic acid sequences precisely in living cells from diverse species. Continued advances in genome editing exponentially broaden our knowledge of human genetics, epigenetics, molecular biology, and pathology. Currently, gene editing-mediated therapies have led to impressive responses in patients with hematological diseases, including sickle cell disease and thalassemia. With the discovery of more efficient, precise and sophisticated gene-editing tools, more therapeutic gene-editing approaches will enter the clinic to treat various diseases, such as acquired immunodeficiency sydrome (AIDS), hematologic malignancies, and even severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These initial successes have spurred the further innovation and development of gene-editing technology. In this review, we will introduce the architecture and mechanism of the current gene-editing tools, including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease-based tools and other protein-based DNA targeting systems, and we summarize the meaningful applications of diverse technologies in preclinical studies, focusing on the establishment of disease models and diagnostic techniques. Finally, we provide a comprehensive overview of clinical information using gene-editing therapeutics for treating various human diseases and emphasize the opportunities and challenges.

Globally, it is estimated that 43 million people are living with human immunodeficiency virus type 1 (HIV-1), and there are more than 600,000 acquired immunodeficiency syndrome (AIDS)-related deaths in 2020. The only way to increase the life expectancy of these patients right now is to use combination antiretroviral therapy (cART) for the lifetime. Due to the integration of the HIV-1 DNA in lymphocytes, the replication of the virus can only be reduced by using antiretroviral drugs. If the drug is stopped, the virus will replicate and reduce the number of lymphocytes. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9-mediated genome editing system has been considered, preventing HIV-1 replication by causing DNA double-stranded breaks (DSBs) or disrupting the integrated virus replication by targeting the provirus. In this study, we utilized the CRISPR/Cas9 without the nuclear localization signal sequence (w/o NLS) system to inhibit the VSV-G-pseudotyped HIV-1 replication by targeting the HIV-1 DNA as a prophylactic method. To this end, we designed a multiplex gRNA (guide RNA) cassette to target the pol, env, and nef/long terminal repeat (nef/LTR) regions of the HIV-1 genome and then cloned it in plasmid expressing no-NLS-Cas9 protein as an all-in-one CRISPR/Cas9 vector. Using HIV-1 pseudovirus transduction into HEK-293T cell lines, our results showed that the CRISPR/Cas9-no NLS system disrupts the pseudotyped HIV-1 DNA and significantly (P-value < 0.0001) decreases the p24 antigen shedding and viral RNA load in cell culture supernatants harvested 48h after virus transduction. Although these results revealed the potential of the CRISPR/Cas9-no NLS nuclease system as a prophylactic strategy against HIV-1 infections, due to inefficient impairments of HIV-1 DNA, further studies are required to enhance its effectiveness and application in clinical practice.

Stem cell therapies are becoming a major topic in biomedical research all over the planet. It may be a viable treatment choice for people suffering from a wide range of illnesses and injuries. It has recently emerged as an extremely intriguing and well-established science and research topic. Expectations have risen due to advancements in therapeutic approaches. Multiple laboratory testing of regulated stem cell culture and derivation is carried out before the formation of stem cells for the use of therapeutic process. Whereas HIV infection is contagious and can last a lifetime. Researchers are still working to develop a comprehensive and effective treatment for HIV and its associated condition, as well as AIDS. HIV propagation is primarily restricted to the immune system, notably T lymphocytes, as well as macrophages. Large numbers of research studies have contributed to a plethora of data about the enigmatic AIDS life cycle. This vast amount of data provides potential targets for AIDS therapies. Currently, stem cell transplantation, along with other procedures, provided novel insights into HIV pathogenesis and offered a glimpse of hope for the development of a viable HIV cure technique. One of its existing focus areas in HIV and AIDS research is to develop a novel therapeutic strategic plan capable of providing life-long complete recovery of HIV and AIDS without regular drug treatment and, inevitably, curative therapy for HIV and AIDS. The current paper tries to address the possibilities for improved stem cell treatments with "bone marrow, Hematopoietic, human umbilical cord mesenchymal, Genetical modifications with CRISPR/Cas9 in combination of stem cells, induced pluripotent stem cells applications" are discussed which are specifically applied in the HIV and AIDS therapeutic management advancement procedures.

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas) system, referred to as CRISPR/Cas system, has attracted significant interest in scientific community due to its great potential in translating into versatile therapeutic tools in biomedical field. For instance, a myriad of studies has demonstrated that the CRISPR/Cas system is capable of detecting various types of viruses, killing antibiotic-resistant bacteria, treating inherited genetic diseases, and providing new strategies for cancer therapy. Furthermore, CRISPR/Cas systems are also exploited as research tools such as genome engineering tool that allows researchers to interrogate the biological roles of unexplored genes or uncover novel functions of known genes. Additionally, the CRISPR/Cas system has been employed to edit the genome of a wide range of eukaryotic, prokaryotic organisms and experimental models, including but not limited to mammalian cells, mice, zebrafish, plants, yeast, and Escherichia coli. The present review mainly focuses on summarizing recent discoveries regarding the type II CRISPR/Cas9 and type VI CRISPR/Cas13a systems to give researchers a glimpse of their potential applications in the biological and biomedical field.