CRISPR/CAS as a Powerful Tool for Human Immunodeficiency Virus Cure: A Review

Abstract

Despite care and the availability of effective antiretroviral treatment, some human immunodeficiency virus (HIV)-infected individuals suffer from neurocognitive disorders associated with HIV (HAND) that significantly affect their quality of life. The different types of HAND can be divided into asymptomatic neurocognitive impairment, mild neurocognitive disorder, and the most severe form known as HIV-associated dementia. Little is known about the mechanisms of HAND, but it is thought to be related to infection of astrocytes, microglial cells, and macrophages in the human brain. The formation of a viral reservoir that lies dormant as a provirus in resting CD4⁺ T lymphocytes and in refuge tissues such as the brain contributes significantly to HIV eradication. In recent years, a new set of tools have emerged: the gene editing based on the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system, which can alter genome segments by insertion, deletion, and replacement and has great therapeutic potential. This technology has been used in research to treat HIV and appears to offer hope for a possible cure for HIV infection and perhaps prevention of HAND. This approach has the potential to directly impact the quality of life of HIV-infected individuals, which is a very important topic to be known and discussed.

Introduction

Combined antiretroviral therapy (cART) is a key strategy to reduce morbidity and mortality from human immunodeficiency virus (HIV)-associated diseases by 42% in ∼73% of infected patients.¹ As of the end of 2020, about 37.6 million people were living with HIV or acquired immunodeficiency syndrome, which remains a global health problem. According to the World Health Organization (www.who.int/hiv/en/), there were ∼1.5 million new infections in 2020. This indicates that current therapeutic strategies are unable to effectively eliminate dormant viral reservoirs and cure HIV infection.¹ One of the disadvantages associated with prolonged treatment of HIV-1 is the high cost of treatment, the possible occurrence of side effects, and the presence of a variety of viral variants that have resistance mutations to different classes of drugs. Recently, research has increasingly focused on novel therapeutic approaches aimed at inhibiting HIV-1 replication and eliminating latent reservoirs.²

HIV reservoirs form in the early phase of infection^3,4 and have been localized primarily in the brain,^5,6 peripheral blood,^7
–9 and lymphoid tissue,^3,10,11 involving mainly resting memory T cells in lymph nodes,^3,10,11 astrocytes,^12,13 microglial cells,^5,13 and macrophages in the brain,^5,14 and resting memory T cells and monocytes^3,7
–9 in peripheral blood. To date, the mechanism of HIV reservoir formation and reactivation of latent cells is unclear. However, there is evidence that HIV provirus is silenced in this reservoir by several factors, including site of integration,^15,16 chromatin status,^17,18 accessibility of transcription factors,^18
–20 and RNA interference.^21
–23

One of the main obstacles is that antiretroviral drugs have suboptimal concentrations in reservoirs, which prevents adequate viral suppression at these sites.^24
–26 Of particular concern, ∼30%–50% of patients currently treated with highly active antiretroviral drugs (cART) have HIV-associated neurologic disorders (HAND).

HAND has previously been found to encompass various neurocognitive impairments at different levels attributable to HIV infection of the brain,^27
–29 and is classified into asymptomatic neurocognitive impairment, minor neurocognitive disorder, and severe HIV-associated dementia (HAD).^30,31 Previous study has recognized the critical role of HAND and its relationship to neuronal resistance to HIV infection as a consequence of disruption of neuronal function and homeostasis.¹³

Astrocytes play a critical role as supporting cells in the human central nervous system (CNS) and have critical functions in both physiological and pathological conditions. They provide a support function for human CNS cells and for part of the blood–brain barrier (BBB). They also form tripartite synapses, release and take up neurotransmitters, and supply energy to neurons. It is known that under pathological conditions they can become dysfunctional and fail to maintain homeostasis.³¹

Neurologic impairment in people living with HIV (PLWHIV) is a major challenge. The neurologic disorders favored by infection range from sensory-motor disturbances to depression and dementia.³² An estimated 70% of HIV-infected patients develop some type of neurologic disorder in the HAART era, including many cases of HAD. Although severe and progressive neurocognitive impairment has become rare in the cART era, the majority of HIV patients worldwide have alterations on formal neurocognitive testing. These symptoms are caused by a variety of disorders that are so specific that diagnosis can be difficult. The literature on HIV infection highlights depletion of CD4⁺ T cells as one of the most important effects on the immune system and its association with nervous system damage. HIV-associated primary dementia, together with opportunistic infections and malignancies, constitutes the so-called neurological manifestations of HIV.³³

In PLWHIV with delayed treatment of HIV infection, opportunistic neurologic infections, meningitis, especially cryptococcal and tuberculous meningitis, toxoplasma encephalitis, and progressive multifocal leukoencephalopathy may occur, among other complications. These complications increase the risk of long-term cognitive impairment. In addition, peripheral neuropathy can affect the quality of life of HIV patients, but it can be reduced if non-neurotoxic ARV treatment is started soon after the diagnosis of infection.³³

Chronic HIV infection is often associated with cognitive decline manifested by loss of attention, learning ability, and executive functions, as well as mood disorders, which significantly affects the quality of life. HIV can cross the BBB as cell-free viral particles or in infected monocytes and T cells, and the presence of viral proteins in the bloodstream stimulates the brain endothelial cells to release cytokines, resulting in persistent inflammation.³³

The cytokines tumor necrosis factor (TNF) and interleukin-1β (IL-1β) activate microglia and astrocytes in the CNS in response to HIV infection, which in turn triggers neuroinflammation leading to the release of neurotoxic substances such as inflammatory mediators and excitatory amino acids. There is a link between the dysfunction and death of neurons and dementia.³⁴

The significant negative impact of HAND, added to other factors such as psychosocial problems and depression, is directly related to treatment adherence and consequently to treatment success and quality of life in these patients.³⁵ Both HAND and depression in PLWHIV may be associated with activation of microglia and astrocytes, release of proinflammatory cytokines, increased permeability of the BBB, and accumulation of free radicals.^36,37

CRISPR/Cas9 Technology

In recent years, the development of gene editing strategies, mainly the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9, has emerged as powerful platforms for DNA modifications and revolutionized molecular precision medicine.³⁸ The story of CRISPR began in the Mediterranean at the University of Alicante, Spain, with a graduate student, Francisco Mojica. He observed a palindromic structure separated by spacers in the microbe archaea. Remarkably, Mojica's observations, made using experiments and bioinformatic tools, led to the characterization of a structure he called “regularly interspaced clustered palindromic repeats,” which later became known as CRISPR.³⁹

Groundbreaking research was carried out by Jinek et al., who showed that a Cas9 DNA endonuclease guided by two RNAs could cleave target DNA in vitro,³⁴ a gene editing system that is a natural defense mechanism in archaea bacteria against viral infections that can be adapted and used in human and other mammalian cells.⁴⁰ Since then, the CRISPR/Cas9 technology has been rapidly developed and made great progress in the field of gene therapy in human CD34⁺ hematopoietic stem cells and progenitor cells.² In 2020, Charpentier and Doudna received the Nobel Prize for their discovery that microbial immune mechanisms can be turned into a toll (CRISPR/Cas9); subsequently, several correlated studies have been attributed to this discovery (Fig. 1).^41,42

FIG. 1.

Time line of the CRISPR/Cas system with important milestones. CRISPR, clustered regularly interspaced palindromic repeats. Adapted from Nidhi et al.⁴²

The CRISPR/Cas9 technology has been used not only to interfere with gene sequences but also to interfere with other biological functions. For example, mutations of the Cas protein in two nuclease domains result in inactive dead Cas9, a site-specific DNA-binding protein used to block gene transcription.² Since its discovery, the CRISPR system has been further developed, improved, and adapted to make the editing and modification of any DNA and RNA molecules increasingly reliable and accurate. This has led to research into the treatment of hereditary and viral diseases, as well as other applications such as the development of molecular imaging.³⁸

Type II CRISPR/Cas9 systems are widely used in genetic engineering due to their ability to facilitate gene editing with remarkable efficiency. In these systems, the Cas9 protein precisely targets the desired DNA region using crRNA and transactivating crRNA. It is able to bind to and cleave almost any nucleic acid site within the target sequence. CRISPR/Cas9 systems can be divided into two types, which are briefly discussed in the following section.³⁸

The CRISPR/Cas9 systems are unusual in that they confer heritable adaptive immunity to the invading genomic material to prokaryotes. The CRISPR genomic locus stores nucleic acid spacer sequences acquired from invading genetic elements, which are then used to guide Cas proteins for targeted removal of foreign intruders. CRISPR/Cas9 systems act at the molecular level through processes such as adaptation, crRNA maturation, and interference, and exhibit tremendous biological variety in many systems. Currently, there are two classes of CRISPR/Cas9 systems, each comprising six types and several subtypes. Class 1 systems (types I, III, and IV) use interference machinery consisting of many Cas proteins, whereas class 2 systems (types II, V, and VI) use a single Cas protein.⁴³

Strategies for using CRISPR/Cas9 to cure HIV infection

Despite the care and treatment of PLWHIV, infection persists due to the formation of viral reservoirs in the early stages of infection. One of the most important aspects of starting treatment after diagnosis is to try to reduce the size of this reservoir, as it is one of the main barriers to curing HIV.⁷ The viral reservoir is dynamic and can persist through mechanisms such as clonal expansion and infection of new cells. Even with the use of cART, there is residual viral replication, which may be related to cell type, type of the provirus, location of integration site allowing potential transcription or silencing, and access to antiretroviral drugs in the tissue.^44,45

In addition to persistent HIV residual replication at certain anatomical sites, there is evidence that defective proviruses can produce transcripts that contribute to a chronic inflammatory state.⁴⁶ As mentioned in this review, one of the tissues in which HIV persists is the brain, which is ideally suited to eliminate HIV as part of a cure and prevent the onset of neurocognitive impairment such as HAND.⁴⁷ When we speak of an HIV cure, we mean remission, that is, control of the virus without cART and its eradication.⁴⁴

To date, several strategies have been explored to cure HIV. One of them is the “Shock and Kill” strategy, in which transcriptional activators (latency reversing agents) are used to activate resting cells and reactivate proviruses for the production of viral particles and proteins.⁴⁸ These drugs can modulate chromatin, activate transcription, or control post-transcriptional processes. In addition to this strategy, cART is used to eliminate the viruses produced.^46,49

Hematopoietic stem cell transplantation is another strategy. It is known that HIV must bind to the CD4⁺ receptor and a coreceptor, mainly chemokine receptor 5 (CCR5), to invade and fuse with the target cell. Homozygous deletion of 32 base pairs (bp) in the CCR5 gene results in resistance to HIV infection.⁵⁰

In this strategy, the patients would have to be eligible for bone marrow transplantation in addition to HIV status, they would have to meet all compatibility criteria based on the leukocyte antigen system (human leukocyte antigen), and the donor cells would have to have the CCR5 deletion, as was the case in the London and Berlin patients. After transplantation, cART was suspended without viral rebound. In the London patient, viral load was not found in the rectum, cecum, or semen and was only positive in some lymphoid tissues but without replication viability. The main difference between the two cases is that the London patient achieved complete remission, whereas the Berlin patient received a second transplant after cancer rebound.^51,52 This strategy would not be considered for all PLWHIV because bone marrow transplantation is considered a high-risk procedure, depends on a compatible donor, and even contains CCR5Δ32 cells.

Block and Lock is a strategy in which the virus can be deactivated by silencing its genome and chromatin with small interfering RNAs and short hairpin RNAs that target conserved regions of the HIV genome. The virus is not eliminated, and patients have a profile similar to individuals with long-term HIV infection without progression without ART, known as elite controllers. They have high counts of CD4⁺ T cells and low or undetectable viral load.^53,54

In the context of HIV cure, genome editing tools have emerged in recent years. These include transcription activator-like effector nucleases (TALENs), which are genetically engineered nucleases that bind to and cleave specific DNA sequences, resulting in insertions/deletions (indels) as a consequence of cellular repair mechanisms. The use of TALENs was initially suggested as a means to treat HIV infection because they alter genes that are important for viral entry into host cells, such as the genes for CCR5, CXCR4, and LEDGF/p75, involved in viral integration. In the virus, TALENs can be used to edit the viral long terminal repeat (LTR). In addition to TALENs, zinc finger nucleases (ZFNs) have also been well studied for disrupting CCR5/CXCR4 in CD4⁺ T lymphocytes.^55,56 Qu et al. developed ZFNs that target the TAR region in the HIV LTR by efficiently cleaving the provirus in the Jurkat T cell lineage.^57,58

The major challenges in gene editing are off-target editing, binding at nonspecific sites that lead to genomic instability and/or disrupt normal gene function, and cytotoxicity and immunogenicity.⁵⁹ In recent years, CRISPR technology has emerged as a way to edit and remove the HIV genome from host cells. The first application of CRISPR/Cas9 to suppress HIV-1 expression in Jurkat cells was performed by Ebina et al.⁶⁰ Hu et al. subsequently used CRISPR/Cas9 to identify specific targets in viral LTRs to remove the complete provirus from myeloid cells that serve as a reservoir for HIV-1.⁶¹ In another study, the authors focused on CCR5 coreceptor editing and used a lentivirus expressing CCR5-directed single-guide RNA (sgRNA) and Cas9 to knock out the CCR5 coreceptor on CD4⁺ T cells.⁶² Holt et al. edited the genes encoding CXCR4 and CCR5 from different lines of CD4⁺ T cells and primary cells.⁵⁸

Kaminski et al. applied the CRISPR technology to remove the viral genome from CD4⁺ T cells, and in another study, they injected vector plasmids expressing Cas9 and various sgRNAs into transgenic mouse models and showed that the HIV provirus could no longer be detected in the spleen, liver, heart, and lung, underscoring that HIV proviruses can be eliminated in vivo using CRISPR.⁶³

Problems with CRISPR gene editing and delivery

Due to their outstanding precision, efficiency, and cost-effectiveness, CRISPR/Cas systems have been widely used. Despite the development of various types of CRISPR/Cas systems, their widespread in vivo application may face obstacles due to off-target effects. To mitigate these effects, work is underway to further develop CRISPR/Cas systems that have higher reliability and accuracy. Several techniques have been used to detect and measure the extent of off-target effects.⁶⁴

CRISPR/Cas9 nucleases are extensively used in genetic engineering in various organisms, including crucial crops. However, it is crucial to address the problem of off-target mutations resulting from these editing techniques. Comparative studies show that the monomeric CRISPR/Cas9 system is more prone to off-target effects compared with the dimeric ZFN and TALEN systems. The dimeric nature of ZFN and TALEN helps identify shorter target sequences, reducing off-target effects.⁶⁵

Off-targets are sequences in the host DNA in which the guide RNA (gRNA) has high homology to the actual target region. Excessive binding and cleavage are common at off-target sites with less mismatched regions. However, several methods are being developed to identify regions with a low off-target probability for precisely constructed gRNA sequences and to minimize the number of mismatches.⁶⁶ The off-target effects of Cas9 were initially studied in human cancer cell lines 67–69 and were found to be remarkably numerous.^67
–69 This may be attributed to the compromised functionality of DNA repair pathways in tumor cells, leading to an elevated incidence of off-target effects.⁶⁵

Several strategies have been widely used to decrease gRNA-dependent off-target base editing,^70,71 for example, incorporation of mutations that increase the DNA specificity of the Cas9 component of base editors (BEs), addition of 5′-guanosine nucleotides to sgRNA,⁷⁰ or delivery of BEs as a ribonucleoprotein (RNP) complex.^70,72,73 Off-target editing by independent gRNA is described below.^70,72,74

Types of Off-Target Effects

So far, three main types of off-target regions have been identified. The first category comprises PAM variants with mismatched or substituted bases in the 5′-NGG-3′ region.^68,75 The second type includes regions within alternative PAMs (5′-NGG-3′) that have indels in comparison with the target DNA or gRNA spacer.⁷⁶ Under these circumstances, the DNA or RNA forms a small bulge with the remaining nucleotides that allows for appropriate hybridization and activation of Cas9. It should be noted, however, that the off-target activities observed at these sites may occasionally exceed the on-target activities. The third type involves cleavage of sequences with distinct PAM (5′-NAG-3′) sites.^68,75

It has been suggested that CRISPR-induced off-target effects can be categorized into two distinct groups. The first category refers to off-target effects that are expected to occur in genomic regions that share significant sequence similarity with the target region. The second category includes unexpected off-target regions of the genome that are unrelated to the intended target.⁷⁷

Mechanism of Off-Target Effects by the CRISPR/Cas System

Accurate genome sequence information is essential for predicting off-target effects. In general, the CRISPR/Cas system requires at least three mismatches within a 20 bp DNA target sequence.^75,78,79 The first step involves the interaction between Cas9 and DNA, which is facilitated by recognition and binding at the PAM site. This allows fusion of the adjacent PAM DNA and thus targeted exploration of potential editing sequences by combining crRNA and forming an R-loop structure.^76,80,81

The pairing and cleavage processes have been shown to have distinct requirements for complementarity within the 20-bp target region. To achieve a highly stable binding, 7–9 bp must match in the closest regions of PAM. Interestingly, studies have shown that four mismatches at the PAM-distal end hinder cleavage, while pairing is still possible.^82,83 The specificity of CRISPR/Cas9 is primarily determined by the sequence of sgRNA seeds located within 10–12 bp directly 5′ from the proximal NGG-PAM region.⁸⁴ Off-target editing occurs when the sgRNA sequence recognizes partial off-seed mismatches instead of target sites.⁸⁵ At these sites, DNA bubbles help bind and cleave the off-target sequences. Partially unwounded or melted DNA helps recruit Cas9 to previously hidden and confounding off-target sites during binding and cleavage.⁸⁶ The off-target editing mechanism by the CRISPR/Cas system is illustrated in Figure 2.

FIG. 2.

Off-target editing engine. CRISPR/Cas9 systems accept at least three mismatches in a 20 bp DNA target sequence. Cas9 can introduce unexpected off-target mutations because sgRNAs recognize DNA sequences with one to a few nucleotide mismatches. (A, B) The connection and PAM recognition mediate the initial Cas9-DNA interaction, which leads to proximal PAM DNA fusion. (C) Binding of the gRNA at the target site initiates the unwinding of the DNA. (D) A stable R loop was formed between the crRNA and the potential target sites. (E) The sgRNA sequence recognizes partial mismatches outside the seed sequence instead of target locations. (F) The off-target edit is generated as a result of mismatch recognition by sgRNA. bp, base pairs; gRNA, guide RNA; sgRNA, single-guide RNA. Adaptated from Manghwar et al.⁶⁴

Major Concerns/Outcomes of Off-Target Effects

CRISPR/Cas systems have remarkable potential for genome editing. However, off-target effects pose a major challenge to host organisms. Off-target effects can lead to chromosomal rearrangements and affect genome sites with limited compatibility. This limitation hinders the widespread application of genome editing in medicine.^87,88 In addition to affecting chromosome stability, off-target effects can lead to loss of functional gene activity, causing various physiological or signaling abnormalities (Fig. 3).^75,89 Therefore, it is critical to design an optimal sgRNA to achieve high targeting accuracy with little or no possibility of off-target effects.⁹⁰ Computational biology is undoubtedly important for the development of bioinformatic tools to identify the best gRNA sequences, which greatly increases the efficiency of CRISPR systems for viral genomes. This approach considers viral diversity with its mutation rates, different genotypes and subtypes, and targets functional regions of these genomes.⁶⁴

FIG. 3.

Top concerns and results of off-target effects. SPR/Cas systems generally support great potential in genome editing, but off-target activity has consequences, not limiting their intended therapeutic applications. (A) CRISPR/Cas9 with a specific sgRNA can sometimes bind and edit at a different location from its target sequence, known as off-target editing. This can result in serious unforeseen events, such as consequence/target inactivation that can result in unwanted phenotypes, or leave cancer-causing oncogene genes occurring in animals. (B) CRISPR/Cas9 that precisely edits its target gene is called target editing. CRISPR/Cas has been used in a wide variety of animals due to its robust on-target editing efficiency. Target editing does not bring the targeted phenotype to the desired ones. Adaptated from Adaptated from Manghwar et al.⁶⁴

CRISPR/Cas system delivery strategies

The biggest challenge to the success of gene therapies is how these systems can reach the target tissue and the correct cell type. Several delivery strategies have been developed over time, and the CRISPR system would not be different. The CRISPR/Cas9 system is delivered as an RNP complex that prevents host genome integration, cellular gene expression problems, RNA degradation, and protein folding.⁹¹ Thus, what makes the CRISPR system efficient is its components and how they are delivered. Several strategies aim to combine efficiency, precision, and safety, including viral vector-based delivery such as adeno-associated viruses (AAVs), lentivirus, and adenoviral vectors (AdVs), while nonviral vectors such as synthetic nanoparticles based on liposomes, polymers, or polypeptides, extracellular vesicles (EV-based delivery), and more recently, exosome-enveloped viral vectors (vexosomes).⁹²

AAVs are nonenveloped single-stranded DNA viruses that are not pathogenic to humans and are not associated with any disease, although 80% of the population is seropositive for these viruses. The use of AAVs as a delivery system has may advantages, including low immunogenicity and cytotoxicity, limited ability to integrate into the host genome, ability to infect dividing or nondividing cells, and the fact that there are multiple serotypes and tropism for different tissues.^93,94 The AAV capsid contains a protein that can lead to immunogenicity by stimulating host immune cells to produce antibodies, as the majority of the population has anti-AAV antibodies, as observed in blood donors. To circumvent this, the antigenic sites were altered to produce a chimeric capsid bypass the host immune response.

Another viral protein of concern was the Rep protein, which is responsible for viral replication, modulation of gene expression and site-specific integration into AAVs. To avoid integration, the Rep protein was removed from the viral genome and integration became inefficient.⁹⁵ Another approach that had to be considered was to limit the AAV cargo (5 kb) because it is a small virus and the CRISPR cargo contains an sgRNA-CAS9 protein (the most commonly used and derived from Streptococcus pyogenes) with a large size.⁹⁶ To address this problem, several strategies were developed. One is to transduce Cas9-expressing cells with AAVs carrying only sgRNAs; another is to cotransduce the cells with two AAVs, one carrying sgRNA and the other Cas9; three AAVs were constructed for delivery of larger systems; yet another strategy was to use a catalytically inactivated Cas9.

Another strategy to solve the problem of cargo size would be to use smaller Cas9 versions, such as Staphylococcus aureus (SaCas9) and Streptococcus thermophiles (St1 Cas9), transported together with the sgRNA in a single vector⁹⁷; Cas13 is an alternative to Cas9 and is a type of RNA-targeting CRISPR effector protein with the function of silencing the expression of a gene.⁹⁸

Lentiviral vectors are an interesting form of delivery with greater cloning capacity than the AAV vector; they package two copies of an RNA genome, making them suitable for CRISPR/Cas9 delivery; they load sgRNA cassettes with a single transfection event; they can be used in dividing or nondividing cells; they have low immunogenicity; they are stable and efficient for permanent endogenous gene expression for random genome integration; and have a low multiplicity of infection ratio.^99,100 Lentiviruses are enveloped single-stranded RNA viruses. After integration into the host genome, cells can produce multiple copies of sgRNAs with low cellular toxicity.

The disadvantage is random integration, which can increase the level of expressed oncogenes.¹⁰¹ To avoid this, a nonintegrating lentivirus vector has been developed by mutating the integrase gene or the LTRs, and has become one of the most promising vectors because it has better packaging capacity and does not cause insertional mutagenesis; however, there is a possibility of off-target effects due to the long duration of sgRNA gene expression.¹⁰² Adenoviruses are nonenveloped double-stranded DNA viruses that do not integrate into the host genome, eliminating the risk of off-target effects and insertional mutagenesis, and have also long been the subject of biological and clinical investigations.¹⁰³

In a preclinical study, AdVs were used to successfully deliver CRISPR/Cas9 into human alpha-1-trypsin in vivo.¹⁰⁴ Recently, a third-generation, high-capacity AdV has been developed with the viral coding genes removed to carry CRISPR/Cas9 cargo in a simple vector.¹⁰⁵ The biggest problem with the AdV is that most people already have antibodies and using the CRISPR system can cause a host immune response. On the contrary, they are safe because they have been studied for a long time and are inexpensive. For this reason, it became the choice for vaccine development during the SARS-CoV-2 pandemic for COVID-19.¹⁰⁶

Advances in nanotechnology have enabled the exploration of nonviral vector systems for CRISPR/Cas9 delivery. Among them, lipid nanoparticles (LNPs) are the most studied, in which amphipathic lipids with hydrophobic tails and hydrophilic head groups self-assemble in an aqueous environment, to form vesicles with a bilayer structure. Nucleic acids have negative charges, and lipids have positive charges, and form a charge–charge complex with sgRNA-Cas9. When they interact with the host cell, they are endocytosed, and this complex prevents the degradation of nucleic acids. Unfortunately, the efficiency of LNPs to package the CRISPR system using plasmids in primary cells and in vivo animal experiments is low, and it is still a challenge to develop efficient and stable delivery systems for various tissues and organs.¹⁰⁷

A positive example was the use of an LNP, the cationic liposome containing Cas9 plasmid, sgRNA and cDNA encoding an enzyme called alpha-l-iduronidase, administered intravenously to knockout mice, which resulted in an improvement in the activity of this enzyme. Deficiency of alpha-l-iduronidase causes a rare disease called mucopolysaccharidosis in humans.¹⁰⁸ One strategy to improve the application of LNPs has been to develop a method to improve the integrity of RNP by adding an ionizable cationic supplement, DOTAP, which requires only small doses and increases the efficiency of delivery to various tissues.¹⁰⁹ Polymer-based nanoparticles have been used, which have a more flexible structure and can release various nucleic acids. The most commonly used for CRISPR/Cas9 delivery are polyethylenimine and chitosan, which can cross the cell membrane by endocytosis, and have lower off-target effects because they protect the CRISPR system from immune reactions and degradation by nucleases.¹¹⁰

Another strategy is that DNA nanostructures are assembled by rolling cycle amplification and can be used for CRISPR/Cas9 RNP delivery.¹¹¹ Gold nanoparticles are widely used nanoparticles because they are inert, biocompatible, and have low toxicity. Moreover, the charge and hydrophilicity of their surface can be easily manipulated. Intracranial delivery of RNA-guided/Cas9 and Cpf1 into the brains of mice by CRISPR-Gold was effective in lowering mGluR5 levels and reversing autism caused by fragile X syndrome. Because CRISPR-Gold technology can precisely edit specific brain cells, it provides an opportunity for the future application of CRISPR technology in various neurogenetic diseases.¹¹²

Currently, EVs have astonishing potential compared with other vectors, as they can deliver different cargoes, such as saccharides, nucleic acids, and proteins. EVs are classified into exosomes, microvesicles, and apoptotic bodies. Exosomes are released from the plasmatic membrane of the cell, fused with endosome-derived microvesicles and released into the plasma. The membrane structure of EVs protects nucleic acids and proteins from serum and immune system endonucleases.¹¹³ In addition, their biocompatibility allows them to easily cross natural biological barriers, such as dense tissues, acidic and enzymatic microenvironments, and the BBB to deliver their cargo.¹¹⁴ When combined with synthetic liposomes, exosomes can be modified in various ways, such as size, and they can transport large cargoes such as CRISPR/Cas9. The genetic material is introduced into the exosome by electroporation, and changes in its structure do not affect its efficiency or cell tropism.¹¹⁵

Artificially engineered exosomes are used for drug, miRNA, and small molecular delivery, and sgRNA-Cas9 can be encapsulated by the exosome and perform gene editing activity within the target cell and reducing the off-target effect.¹¹⁶ Vexosomes are a novel gene delivery strategy and an exosome-enveloped viral vector with multicellular and tissue tropism that is nearly nonimmunogenic. They are produced by infecting cells with plasmids encoding the viral genome, which may be AAVs, so that the cytoplasm is enriched with viral genomes and proteins recognized by the plasma membrane and endosomal, and phagosomal receptors. Vexosomes have not yet been used for delivery of the CRISPR/Cas system,¹¹⁶ although their potential to combine the characteristics of viral vectors and EVs may be a promising tool in the near future.⁹²

CRISPR and HAND

Microglia are the principal immune-responsive cell type in the CNS and play a critical role in neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Modulation of microglial cell activation may provide a safe and effective approach for the treatment of these neurodegenerative diseases. This is particularly important because no drugs can act simultaneously on multiple cells in a cell-nonspecific manner. The CRISPR/Cas system has shown promising results in various neurological diseases. However, there are concerns about off-target mutations and immunogenicity in gene editing, highlighting the need to explore precise delivery methods of this tool to the intended CNS target.¹¹⁷ Luo et al. have well described how the CRISPR/Cas9 technology has altered in Parkinson's disease studies through apoptotic pathways associated with neurodegenerative disease processes.¹¹⁸

Raikwar et al. note that CRISPR/Cas9 editing holds tremendous potential for deciphering the complex molecular pathways linked to neuroinflammation and neurodegeneration in various neurodegenerative diseases.¹¹⁹ The adaptability of the CRISPR/Cas9 system extends to other cell types in the CNS, including neurons and oligodendrocytes. This remarkable versatility suggests its potential application as a therapeutic method for treating neurodegenerative and neuroinflammatory diseases. Neuroinflammation, which is closely related to various neurological diseases and disorders such as brain trauma, ischemia, viral infections, and neurodegeneration, emphasizes the urgent need for discovering effective treatments.¹²⁰

CRISPR/Cas9 holds great promise as a strategy for manipulating the amyloid precursor protein (APP) and its cleavage process in the CNS. To increase the production of the neuroprotective cleaved form of APP, researchers utilized CRISPR/Cas9 to induce modifications in the C-terminus of APP. The findings of the study revealed a significant decrease in total APP, while the protective variant of APP demonstrated a significant increase when a specific delivery method was used. These results were observed in both wild-type-induced pluripotent stem cells and cells harboring Alzheimer's disease mutations.¹²⁰ The CRISPR/Cas9 system, reported by Qin et al., has opened new avenues for altering the expression of genes implicated in neuroinflammation and chronic pain. The potential of this method to alter gene expression in such circumstances is promising.¹²¹

Extensive research over the past two decades on the relationship between HIV and the CNS has highlighted the importance of including the brain in efforts to cure HIV.^47,122 Furthermore, there is an urgent need for innovative therapeutic techniques that target HIV in brain cells.^123,124 One promising approach to preventing HIV expression is the selective eradication of proviruses using genome editing tools such as CRISPR/Cas9.¹²⁵ Several investigations have provided compelling evidence of the potential of the CRISPR/Cas9 system to disruptively alter the HIV-1 provirus,^{60,61,63,126,127} and thereby prevent its expression.¹²⁸

The brain is not only populated by macrophages and astrocytes susceptible to HIV-1, but is also compartmentalized and protected by the BBB, so that cells and biomarkers can selectively transported. Furthermore, HAND are still prevalent in our patients despite the absence of blood viremia, and the use of CRISPR would be one approach to remove HIV from brain cells such as astrocytes, glia, and neurons.¹²⁹

tat and ltr genes have been widely used in in vitro experiments with glial cell cultures and different CRISPR/Cas strategies to prevent or cure HAND. As mentioned in this review, HIV-1 integrates its genetic material into the host genome and over time forms an extensive reservoir of latent cells with inactive viral production, which is the main obstacle to curing HIV. This is no different in the CNS, where HIV-1 infects and remains latent in brain glial cells such as astrocytes and microglia, which are closely associated with the occurrence of HAND.⁵

The presence of HIV-1 in the CNS causes dysregulation of neuronal function and toxicity, and glial cells by direct or indirect mechanisms, many of which are mainly related to the tat and ltr genes.¹³⁰ Therefore, these genes have been widely used to develop various CRISPR/CAS in vitro strategies to remove the provirus or disrupt viral replication in CNS cells.⁶⁰

TAT protein, a transcriptional transactivator, is detected in the cerebrospinal fluid of HIV-positive patients on cART with undetectable viral load in plasma at levels that vary over time.¹³⁰ The effects on CNS dysregulation are caused by the TAT protein through direct damage to glial cells by the production of viral particles and interaction with the ltr gene and cellular machinery; cytotoxicity and cell loss through continuous immune activation with the production of cytokines and proinflammatory molecules (glial cells and neurons); indirect damage by altering the expression of important proteins, such as claudins and occludins, which are responsible for the integrity of BBB and cause an increase in permeability; TAT internalizes into neurons interacts directly with the p53 protein causing loss of neurons by apoptosis.¹³¹

The LTR, located at the ends of the integrated HIV provirus, has become a promising target for excision of the HIV genome from the host cell. It is responsible for initiating transcriptional by interacting with the viral TAT protein as well as cellular transcription factors to produce viral particles. Furthermore, the LTR contains cis-regulatory elements that bind to transcription factors involved in the regulation of HIV transcription and play an important role in viral latency. The CNS has a single LTR promoter that contains mutations in the specificity protein 1 motif, which promotes recruitment of HDAC1 to the LTR and results in methylation directly adjacent to the two nuclear factor kappa B (NF-κB) binding sites, thereby placing the virus in a quiescent state.¹³²

While various strategies have been developed to cure HIV, the need for complex discussions about ethical issues has increased to ensure that gene therapy research, which includes CRISPR, is validated, accepted, and safe for humans. This is because of strategies such as allogeneic hematopoietic cell transplantation for people with HIV and acute myeloid leukemia, cART interruption, and gene manipulation. Indeed, the risk–benefit ratio must be weighed, always considering the minimal risk to the patient.¹³³

The CRISPR tool raises a number of questions that need to be addressed. Leading research groups are currently focused on improving the efficacy and precision of delivery systems, as well as developing bioinformatic tools to identify an ever-widening range of potential off-target effects. When attempting to specifically target a viral genome such as HIV-1, within the human genome, numerous obstacles must be overcome. These hurdles arise from the substantial size of the viral genome, as measured by the number of base pairs, and from its well-organized packaging with nuclear proteins. Currently, several research disciplines are actively working to expand the repertoire of tools for finding an HIV cure. Among these tools, the CRISPR system is undoubtedly promising.

Footnotes

Authors' Contributions

S.V.K.: conceptualization and writing—original draft. W.D.: writing—original draft and review and editing. S.S.S.: writing—review and editing and visualization. V.A.F.: writing—original draft and review and editing. I.N.B.: review and editing. J.C.: writing—conceptualization, review and editing, and supervision.

Consent for Publication

Consent for publication was obtained from all authors.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by grant #2018/07239-2 from the São Paulo Research Foundation (FAPESP), National Council of Scientific and Technological Development (CNPq) scholarship to Jorge Casseb: 30127/2019-0, and Foundation of Faculty of Medicine.

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