Accepted Article Preview: Published ahead of advance online publication

Current application of CRISPR/Cas9 gene-editing technique to

eradication of HIV/AIDS

Z Huang, A Tomitaka, A Raymond, M Nair

Cite this article as: Z Huang, A Tomitaka, A Raymond, M Nair, Current

application of CRISPR/Cas9 gene-editing technique to eradication of HIV/AIDS,
Gene Therapy accepted article preview 4 May 2017; doi: 10.1038/gt.2017.35.

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Received 25 August 2016; revised 25 January 2017; accepted 1 February 2017;

Accepted article preview online 4 May 2017

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Current application of CRISPR/Cas9 gene-editing technique to eradication of HIV/AIDS

Zaohua Huang*1, Asahi Tomitaka1, Andrea Raymond1, Madhavan Nair1*

1 Department of Immunology, Institute of NeuroImmune Pharmacology, Center for Personalized

Nanomedicine, Herbert Wertheim College of Medicine, Florida International University, 11200 SW 8th

Street, Miami, Florida 33199

Keywords: HIV, AIDS, CRISPR/Cas9, gene therapy

*Corresponding Author:

Madhavan Nair
Department of Immunology
Institute of NeuroImmune Pharmacology
Herbert Wertheim College of Medicine
Florida International University
11200 SW 8th Street,
Miami, FL 33199, USA.
Tel: 1-305-348-1493
Fax: 1-305-348-7440
E-mail address: nairm@fiu.edu

Zaohua Huang
Department of Immunology
Institute of NeuroImmune Pharmacology
Herbert Wertheim College of Medicine
Florida International University
11200 SW 8th Street,
Miami, FL 33199, USA.
Tel: 1-305-348-4857
Fax: 1-305-348-7440
E-mail address: zahuan@fiu.edu

© 2017 Macmillan Publishers Limited. All rights reserved.

Abstract:

Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) remains a major

health hazard despite significant advances in prevention and treatment of HIV infection. The major

reason for the persistence of HIV/AIDS is the inability of existing treatments to clear or eradicate the

multiple HIV reservoirs that exist in the human body. To suppress the virus replication and rebound,

HIV/AIDS patients must take life-long antiviral medications. The clustered regularly interspaced

palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is an emerging gene-editing

technique with the potential to eliminate or disrupt HIV-integrated genomes or HIV-infected cells from

multiple HIV reservoirs, which could result in the complete cure of HIV/AIDS. Encouraging progress has

already been reported for the application of the CRISPR/Cas9 technique to HIV/AIDS treatment and

prevention, both in vitro in human patient cells and in vivo in animal model experiments. In this review,

we will summarize the most recent progress in the application of the CRISPR/Cas9 gene-editing

technique to HIV/AIDS therapy and elimination. Future directions and trends of such applications are

also discussed.

© 2017 Macmillan Publishers Limited. All rights reserved.

Introduction

Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) is one of the most

severe pandemic diseases in modern history. In 2015, according to the World Health Organization

(WHO), about 36.7 million people worldwide were living with HIV, and roughly 2.2 million people died of

AIDS, while more than 2.1 million new patients were diagnosed with HIV infection. Of all people

diagnosed with HIV infections, it is estimated that only 17 million (46%) are on antiretroviral therapy

(WHO). With breakthroughs in HIV/AIDS prevention, diagnosis, and treatment, the morbidity and

mortality of HIV/AIDS has decreased significantly. However, HIV/AIDS remains an incurable, chronic

disease due to the multiple HIV latent reservoirs in patients’ bodies. These reservoirs primarily contain

very small numbers (for example, about 1 per million memory CD4+ T cells) of latently infected cells (1-4)

which are located in the brain (5-7), peripheral blood (2, 3, 8, 9), lymphoid tissue (1, 10, 11),

gastrointestinal tract (10, 11) , and other locations (12, 13). Complicated mechanisms underlie the

persistence of HIV reservoirs, including the stability of reservoir cells (4); homeostatic proliferation of

reservoir cells (14); replenishment of reservoir cells (15); and low levels of anti-viral drug penetration

(16-18). Moreover, even under antiretroviral therapy (ART), about 30 to 50% of AIDS patients eventually

develop HIV-associated neurological disorders (HAND) (19, 20), which are cognitive, motor and/or

behavioral impairments caused by HIV infection in the brain (6, 20-23). HAND can further be grouped

into asymptomatic neurocognitive impairment (ANI), minor neurocognitive disorder (MND), and the

most severe r, HIV-associated dementia (HAD) (21). The mechanism of HAND remains to be elucidated,

but it is generally accepted that HAND is tightly correlated with HIV infection of astrocytes (22, 24-26),

microglial cells (5, 26), and macrophages (5, 6) in the human brain. Neurons are believed to be resistant

to HIV infection; however, the neurotoxic products released from HIV-infected brain cells significantly

dysregulate neuronal function and homeostasis (26).

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The ultimate cure for HIV/AIDS will be the removal or disruption of integrated HIV provirus from latently

infected cells or the elimination of these latent cells completely. However, until recently, gene therapy

for HIV/AIDS has progressed very slowly. The breakthrough of gene-editing technology raises the hope

for eradication of HIV as the provirus can be removed from the host cell genome through the newly

developed technique of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-

associated protein 9 (Cas9) gene-editing (27-30). Furthermore, CRISPR/Cas9 system can be utilized to

induce apoptosis in reservoir cells (31, 32). Originally an adaptive immune response mechanism in

bacteria (33, 34), the CRISPR/Cas9 system has emerged as a popular gene-editing system with proven

efficiency in mammalian cells. As early as 2013, soon after the CRISPR/Cas9 technique emerged, its

potential applications for treating HIV/AIDS were considered (28). This mini-review addresses the use of

the CRISPR/Cas9 system as a powerful gene-editing tool to delete or disrupt HIV provirus from latently

infected cell genomes or to knock out CXCR4 and CCR5 receptors for HIV infection treatment and

prevention. The elimination or disruption of HIV provirus in reservoir cells or the elimination of reservoir

cells may result in a cure for HIV/AIDS.

Overview of HIV productive and latent infection

HIV is a retrovirus that belongs to the lentivirus genus. Its DNA genome has two long terminal repeat

(LTR) structures flanking the whole virus genome, which consists of nine overlapping genes: gag, pol,

env, tat, rev, nef, vif, vpr, vpu (Figure 1A). HIV is the causative agent of AIDS in human patients, and it

can be transmitted through contact with infected blood or other body fluids through broken skin or

mucous membranes. The two most common ways to spread HIV/AIDS are unprotected sex and needle

sharing for intravenous drug injection.

HIV targets CD4+ human immune cells, including T cells, dendritic cells, and monocytes, as well as cells

within the central nervous system (CNS), such as perivascular macrophages, astrocytes, and microglial

© 2017 Macmillan Publishers Limited. All rights reserved.

cells. The life cycle of HIV is quite complex (Figure 1B, C). HIV establishes two types of infection in target

cells – active infection (Figure 1B) or latent infection (Figure 1C). Active infection occurs in most cells

while latent infection occurs in much fewer cells (1, 2) and at very early stages of HIV infection (9, 35). In

active infection, HIV provirus is active and HIV virus particles are actively replicated; and the infected

cells continuously release viral progeny; while in latent infection, HIV provirus is transcriptionally

silenced and no viral progeny is produced. However, under certain conditions, such as normal

immunologic responses to various recall antigens or routine vaccination (36), or the induction of

cytokines (2, 35), latently infected cells are reactivated to produce virus (2); subsequently, a pool of new

latent cells are generated by these reactivated latent cells (35). These reservoirs ultimately hinder

HIV/AIDS eradication. The mechanisms of how latent HIV infection occurs remain elusive, but evidence

suggests that the establishment of an HIV reservoir is related to the following factors: HIV provirus

integration site (37-39); chromatin environment (40, 41); accessibility of transcription factors (41-43);

and RNA interference (44-46). Latently infected cells are long-lived and mainly reside in reservoir sites

where antiviral drugs may not penetrate, such as the brain (5-7), lymphoid tissue (1, 10, 11), and

gastrointestinal tract (10, 11). Latently infected cell populations include memory CD4+ T cells (1-4, 10),

macrophages (5, 6, 11), microglial cells (5, 26) and astrocytes (22, 24-26). The establishment of an HIV

reservoir occurs soon after infection, and early application of anti-viral drugs typically only diminishes

rather than eliminates the reservoir (9, 35).

To eliminate the viral reservoirs, current “shock and kill” efforts seek to reactivate the latent reservoirs

(the shock) and destroy the reactivated cells (the kill) through an intensifying combination of

antiretroviral therapy (cART) and host immune responses (47). Research efforts have shown that using

SAHA, a histone deacetylase inhibitor, in conjunction with cART reactivates latent HIV in infected CD4+ T

cells, making these cells susceptible to specific CD8 T cell killing in vitro (48, 49). However, none of the

clinical studies demonstrated a significant therapeutic effect as measured by HIV DNA or quantitative

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viral outgrowth assay, which means they are ineffective (48, 49). Now “shock and kill” efforts have

expanded to include testing and optimizing an increasing number of latency-reversing compounds

combined with host immunomodulatory strategies (47). The setbacks of “shock and kill” have

encouraged researchers to look for alternative approaches to eradicate HIV reservoirs. One approach to

complete HIV eradication would involve the actual removal or disruption of the provirus from HIV

reservoir cells. This type of gene therapy approach has recently been investigated using CRISPR/Cas9

gene-editing system with promising results (27-30).

Overview of CRISPR/Cas9 Technology

The ultimate goal of gene-editing is gene repair. Several gene-editing technologies recently

demonstrated strong potential for therapeutic uses. These technologies include zinc-finger nucleases

(ZFNs) (50-52), transcription activator–like effector nucleases (TALENs) (53-55), and CRISPR/Cas9 system

(34, 56-59). In principle, ZFNs and TALENs fuse DNA nuclease and DNA binding domains together to

break DNA at specific loci, while Cas9 nucleases work with guide RNA to break DNA at specific loci. Both

ZFNs and TALENs techniques have significant applications and studies in HIV/AIDS prevention and

therapy (60-63). However, this review will be focusing solely on the CRISPR/Cas9 system due to the

limited scope.

Compared to the initial CRISPR/Cas9 system in bacteria, the current CRISPR/Cas9 system has been

simplified, modified and adapted for mammalian genome editing and bioengineered for better nucleus

localization and mammalian cell expression (57, 59). The current CRISPR/Cas9 system includes a single

guide RNA (sgRNA) consists of crRNA and tracRNA (57, 59), and Cas9 which excises the target DNA and

sgRNAs which guide Cas9 to the target sites (57, 59). Cas9 is a nuclease of 1368 amino acids with two

nuclease activity domains named HNH (residues 775–908) and RuvC (residues 1–59, 718–769, and 909–

1098) (59, 64-66) (Figure 2A). Each domain can cleave a DNA strand directed by a sgRNA complementary

© 2017 Macmillan Publishers Limited. All rights reserved.

to the target DNA sequence (generally 20 nucleotides long). The prerequisite to be a target sequence is

the presence of a NGG sequence (N: any nucleotides; G: guanine nucleotide, also called protospacer

adjacent motifs (PAMs)) downstream (3’ end) of the target site (59, 64-66). In the nucleus, Cas9, sgRNA

and target DNA form a complex, then HNH and RuvC domains each cleave a DNA strand (59, 64-66)

(Figure 2B). The double-strand DNA breaks are consequently repaired by two different approaches. One

is non-homologous end-joining (NHEJ) when there is no template and the other is homology directed

repair (HDR) when there is a homologous nucleotide template present such as ssDNA or dsDNA. This

template must possess homology sequences (arms) to the two regions that flank the Cas9 excision site.

If the two homologous arms flank a correct sequence which can be used as a template to “repair” the

genomic sequences (59, 67). NHEJ usually results in an insertion or deletion in a DNA sequence, in total

called indel, while HDR results in correct repair as directed by the template (59, 67). CRISPR/Cas9 system

can also be used in biological functions other than gene-editing. For example, catalytically inactive Cas9

(dCas9) has no active nuclease domains and is often applied to be a site-specific DNA binding factor (68).

The fusion protein of dCas9 and transcription activator or repressor domain are often used to regulate

gene expressions (59, 68). In summary, CRISPR/Cas9 system is a full developed multifunction gene-

editing and regulating system which has unlimited potential (59, 67).

Application of CRISPR/Cas9 system to HIV/AIDS prevention and treatments

The first attempt to apply CRISPR/Cas9 system to HIV/AIDS eradication was conducted in Japan in 2013.

Ebina et al. (28) tried to eliminate HIV provirus from T cells by targeting the LTR region of HIV provirus.

CRISPR/Cas9 system was applied to target TAR sequence of the R region and NF-κB binding sequence in

the U3 region. The indel formation by CRISPR/Cas9 cleavage of TAR region is more effective to disrupt

provirus transcription and replication (28). The indel formation by CRISPR/Cas9 cleavage of LTR resulted

in inhibition of active provirus expression and decreased reactivation of latent provirus (28).

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For HIV prevention, many investigators focus on applying the CRISPR/Cas9 technique to the deletion or

disruption of the HIV receptors’ expression on human cells. Since CD4 is the essential primary receptor

in human T cells and is indispensable for normal cell functions, HIV secondary receptors of CXCR4 and

CCR5 are often candidates for the deletion and disruption by CRISPR/Cas9 technique. T cell genome

engineering holds great promise for cell-based therapies for HIV eradication due to the rapid

development of gene-editing. Hou et al. (69) efficiently disrupted the CXCR4 gene with CRISPR/Cas9

technology in human primary CD4+ T cells. Ten sites within the CXCR4 gene were targeted and CXCR4

expression decreased by approximately 30% as confirmed by Western blot (69). The engineered T cells

showed resistance to HIV infection and p24 production reduced over 60% (69). More importantly, this

technology indicated high specificity and no off-target effects, while cell division and propagation

remained normal (69). Schumann et al. (70) used Cas9/sgRNA ribonucleoproteins (Cas9 RNPs) to ablate

CXCR4 gene expression in human T cells, and about 40% of cells lost surface expression of CXCR4. When

paired with repair template, Cas9 RNPs produced a desired genome modification in human primary T

cells (70). Deep sequencing confirmed that Cas9 RNPs achieved about 20% efficiency of knock-in

genome modifications (70). Because of the success of the Berlin Patient case, Ye et al. (71) tried to

produce CCR5Δ32 induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 system. Close to 100%

efficiency was achieved for homologous recombination of a single allele and up to 33% of double alleles

(71). As expected, monocytes and macrophages derived from such engineered iPSCs were found to be

resistant to HIV infection (71). Wang et al. (72) applied CRISPR/Cas9 technology to block CCR5

expression in human CD4+ cells. Lentiviral vectors were used to target three sites of the CCR5 gene (72).

The disruption ratio of CCR5 was high, and those cells were resistant to R5 tropic HIV with negligible off-

target effect (72). However, attempts to disrupt CCR5 gene in human primary T cells with the same

approach failed for unknown reasons (72). In another study, Li et al. (73) applied adenoviral vectors to

deliver CRISPR/Cas9 system to target multiple sites of the fourth exon of CCR5 gene to block CCR5

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expression. First, the TZM-b1 cell line was tested and two important target sites which knockout over 60%

of the gene were identified. Secondly, the results were confirmed in a CHO and a human T cell lines.

Thirdly, CRISPR/Cas9 technique conferred TZM-B1 cell the resistance to HIV infection. Finally, a chimeric

Ad5/F35 adenoviral vector was used to deliver the CRISPR/Cas9 system to human primary CD4+ T cells

to disrupt the CCR5 gene which provided the primary T-cells the resistance to HIV infection (73).

Besides the deletion or disruption of HIV receptors on human cells, many scientists have applied the

CRISPR/Cas9 technique to remove or disrupt HIV provirus in latently infected human cells (Figure 3).

The Drexel Medicine CNS AIDS Research and Eradication Study (CARES) cohort studies (27)

demonstrated the following: 1. The proviral LTR sequences within the peripheral blood mononuclear

cells (PBMC) decreased variations every year with ART; 2. HIV LTR sequences continued genetic changes

for a minimum of 6 years even with effective suppressive ART; 3. Next generation sequencing (NGS)

analysis found that a regimen of sgRNAs could be designed to target all known HIV quasispecies in 50%

of the analyzed patients; and 4. HAART therapy might reduce the required sgRNA scale to eradicate

provirus. For the first time, the study demonstrated the feasibility of HIV provirus elimination with

CRISPR/Cas9 technology in a single patient (27). Zhu et al. (74) targeted ten sites of the HIV provirus to

eradicate HIV DNA from Jukat cell genome. These sites include three within LTR region, five within pol

gene, and two within rev gene (74). The second exon of rev ranked as the best targeting site, and the

targeting at this site caused a 20-fold reduction in HIV production (74). To eradicate the latent HIV

reservoirs and achieve a complete cure of HIV/AIDS, the “shock and kill" strategy is often applied to

reactivate the latent HIV reservoirs and then kill those activated reservoir cells by ART (47-49). However,

the efficiency, specificity and toxicity of current drugs turned out to be disappointing (47-49). Now

CRISPR/Cas9 system is utilized to execute the “shock and kill" strategy, in detail, catalytically deficient

Cas9 (dCas9)-transcription activator fusion protein/sgRNA is applied to specifically activate HIV latent

reservoirs (31, 32). With this technique, Zhang et al.(32) targeted 20 sites within the LTR U3 region of

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HIV provirus and found that the two best activation sites are at or near the NF-κB binding site. This

targeted activation potently reactivated several HIV cell lines, including HIV-1 latent TZM-bI epithelial,

Jurkat T cells and CHME5 microglial cells (32). Moreover, the reactivation resulted in cell death of Jurkat

T cells and CHME5 microglial cells due to the accumulation of viral proteins of TAT, NEF, REV, etc.(32).

Almost at the same time, similarly, Saayman et al. (31) targeted 23 sites in the LTR U3 region of the HIV

provirus and identified the best activation site as at near the NF-κB binding sites. The targeting system

worked in many different in vitro latent T cell models to reactivate provirus, and these reactivations

were consistent and more efficient than latency breaking reagents, such as prostratin and SAHA (31).

More importantly, the activation was specific to the provirus while latency breaking reagents caused

widespread side effects. In addition, the reactivation also blocked the NF-κB reactivation (31). Hu et al.

(29) tried using the CRISPR/Cas9 system to eradicate HIV provirus in microglial cells, pro-monocytes, and

T cells. The LTR U3 region was tested for four sites individually or in combination. It turned out that two

sites combined approach achieved the best eradication results (29). Furthermore, no off-target effects

were found, and the stable expression of Cas9 and sgRNA conferred cells with the ability to resist new

HIV infections (29). Kaminski et al.(30) applied a similar approach to remove the entire HIV provirus

from latently infected human CD4 T cells. No off-target effects were found and cell health parameters

were not affected (30). The persistent expression of Cas9 and sgRNA protected the human CD4 T cells

from new HIV infection (30). The delivery of Cas9 and sgRNA using lentivirus vector could reduce HIV

replication in HIV-infected primary human T cells and decrease viral load in ex vivo patient CD4+ T cells

(30). Kaminski et al. (75) has also applied a recombinant adeno-associated virus 9 (rAAV9) vector

expressing saCas9 (a smaller version of Cas9 derived from Staphylococcus aureus to fit AAV vectors) and

sgRNAs to remove integrated HIV DNA in a mouse transgenic model. A 978 bp DNA fragment was

removed from integrated HIV DNA from lymphocytes and cells in multiple organs, including brain, heart,

kidney, liver, lung, and spleen (75). Similar results were found in transgenic rat experiments (75). This is

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the first report of such studies in transgenic animal models. These encouraging progress demonstrated

the great promise of CRISPR/Cas9 technique in eradication of HIV/AIDS. But there were scientific

findings that pointed out some pitfalls of CRISPR/Cas9 application to HIV/AIDS eradication. Wang et al.

(76) demonstrated that Cas9/sgRNA can inhibit HIV replication initially, but soon HIV-1 escape variants

were produced due to the NHEJ repair, and the escape variants contained mutations around the Cas9

cleavage sites. Interestingly, the more conservative the Cas9/gRNA targeting sequences were , the

longer it took for the HIV escape variants to appear (76). In another research group, Wang et al. (77)

found that: CRISPR/Cas9 could derive mutant HIV which could resist Cas9 and sgRNA reagents due to

the host cell DNA repair. Yoder et al. (78) recently reported research results with similar data and

conclusions. These negative findings suggest the need for careful design and cautious evaluations of the

application of the CRISPR/Cas9 technique to HIV/AIDS eradication. Multiplex gRNAs and several excision

sites of a provirus may be necessary to forever eliminate or disrupt HIV provirus in reservoir cells.

CRISPR/Cas9 System Delivery Approach

The major challenge of curing HIV/AIDS lies in the persistence of multiple reservoirs in several drug-

inaccessible parts of the human body. Moreover, while current HIV/AIDS patients can take medications

to suppress symptoms, the side effects of these drugs may contribute to high morbidity in these

patients. The complete cure is the elimination or disruption of HIV provirus from the patient’s reservoir

cells’ genomes or the elimination of patient reservoir cells. As stated above, gene-editing technology

makes it possible to manipulate DNA at the cell genomic level. Since its discovery, CRISPPR/Cas9 system

has been a premier tool in gene-editing. As mentioned before, many experimental efforts and clinical

trials are underway for the application of the CRISPR/Cas9 system for eradication of HIV/AIDS. The

most substantive progress to date has been reported are ex vivo studies. Future efforts will be directed

towards efficient delivery of CRISPR/Cas9 system components to latently infected cells.

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Currently, the delivery methods of CRISPR/Cas9 system are divided into viral delivery and non-viral

delivery. The CRISPR/Cas9 system is very flexible and can be delivered in the form of DNA, RNA, or

protein/RNA complex. The most popular approach is the DNA form as one or several plasmids. In the

viral delivery methods, lentivirus (29, 79, 80), baculovirus (81), and recombinant adeno associated virus

(rAAV) have been tried (75, 80). Among these virus vectors, rAAV has the advantages of high safety,

flexible administration routes and low or no immune-response, but it has the disadvantage of limited

cargo size. The major administration approaches of rAAV include stereotactic injection, intranasal and

intratracheal administration, intravenous injection, intraperitoneal Injection, and intramuscular Injection.

In the non-viral delivery approach, vectors such as cationic polymer polyethyleneimine (PEI) (82),

liposomes (58, 80), lipid nanoparticles (83, 84), virus-like particles derived from bacteriophage (85), and

self-assembled DNA nanoparticles (86) have been applied for the delivery of the CRISPR/Cas9 system.

Since viral and non-viral approaches have relative advantages and disadvantages, some investigators

started trying the combined approach. For example, lipid nanoparticle-mediated delivery of Cas9 mRNA

has been combined with adeno-associated viruses encoding a sgRNA and a repair template (87). This

delivery strategy successfully repaired a disease gene in an adult mouse model (87).

No matter which delivery strategy is being used, the emphases are biocompatible and targetable

carriers, controllable reagents release, and low-invasive administration. As for HIV/AIDS eradication, in

order to establish more efficient evaluation methods, HIV/AIDS primate models will be the center of the

next phase of study.

Drug brain delivery is the key for the eradication of the HIV reservoir in patients’ CNS systems. The

obstacle for drug brain delivery of the CRISPR/Cas9 system is the existence of the blood brain barrier

(BBB). The tight junction between capillary endothelial cells block transportation of large molecules and

only limited molecules with molecular weight smaller than 400 Da and appropriate lipophilicity can cross.

All large molecules and most of the small molecules cannot cross the BBB. Great efforts have been

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made to improve BBB permeability for therapeutic agents for the treatment of various brain diseases

and HAND. However, currently available procedures are mainly invasive, including

intracerebroventricular (ICV) infusion, intra-cerebral injection. Although these approaches have shown

successful disruption of the BBB, the invasiveness of the technique and the risk of damage to the brain

remain significant concerns. To overcome these problems, the use of nanomaterials as drug delivery

carriers to the brain has been gaining appreciation.

The most popular approach for brain targeting using nanomaterials is receptor-mediated transcytosis

(Figure 4). The endothelial cells which form the BBB are known to express receptors including the

transferrin receptor and insulin receptor (88-90). Ligand functionalized nanoparticles can promote

efficient brain delivery through the binding of ligands to the receptors on endothelial cells. Various

nanoparticles have been developed for brain targeting using this receptor mediated transcytosis, such as

transferrin (91, 92), lactoferrin (93, 94), or monoclonal antibody conjugated liposomes (93), polymer

nanoparticles (95), gold nanoparticles (91), and iron oxide nanoparticles (94, 96).

Magnetic targeting is another approach to improve BBB transmigration of nanoparticles (Figure 4). Nair

et al. (92, 97-100) have made important progress in brain delivery using magnetic nanoparticles and

magneto-electric nanoparticles. Magneto-electric nanoparticles have been used as carrier molecules

which could cross the BBB and mediate controlled release of the loaded drugs. These nanoparticles

were able to cross the BBB using magnetic force induced on the nanoparticles by the magnetic field

gradient (97, 98). To date, these nanoparticles have not been applied to the delivery of the CRISPR/Cas9

system nor have they been fully tested in animal models. However, the potential for these nanoparticles

to deliver CRISPR/Cas9 system and target HIV reservoir in brain seems high due to the nanoparticles’

capacities of specific targeting, crossing the BBB, constant or controlled release and non-invasive

application.

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Conclusion and Future Directions

For the future of applying CRISPR/Cas9 technique to eradicate HIV/AIDS reservoirs, safety, efficiency and

specificity will be the main emphases. Research efforts using the CRISPR/Cas9 technique will now be

focused on primate models and phase I clinical trials. From the point of this review, the CRISPR/Cas9

technique holds great promise for the eradication of HIV/AIDS. Since the FDA has already approved use

of CRISPR/Cas9 technique in several clinical trials, it is surely not long before the CRISPR/Cas9 system

can be applied for clinical therapeutics and prevention on HIV/AIDS.

Conflict of Interest Statement

The authors declare that the manuscript was conducted in the absence of any commercial or financial

relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by grants (R01DA037838 and R01DA034547) from National Institutes of Health,

US. We also thank Maureen Pelham and Courtney Myhr for their help in English editing.

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Figure 1: HIV life cycle

1A: HIV DNA genome HIV-1 DNA genome structure is illustrated above and each gene is drawn in ratio.

The viral genome is approximately 10 kb long and contains nine viral genes. All the viral genes’ locations

are identified. In addition, tat and rev have two exons. TAT protein is expressed in a minor form (72

amino acids) and a major forms (86 amino acids), while REV protein is expressed as one form of 116

amino acids. Genes of gag and env encode structural proteins; gene of pol encodes enzymes and all

other genes encode regulatory proteins. 1B. HIV active infection: In this illustration, HIV life cycle is

divided into seven stages: Binding, fusion and entry (1 and 2): HIV binds to immune cell surface

receptors, including CD4 and CXCR4 or CD4 and CCR5. The binding causes conformation changes and

results in the membrane fusion between HIV and cell membrane. HIV enters the cell, disintegrates and

releases the viral RNA and enzymes. Reverse transcription (3): HIV viral reverse transcriptase converting

viral RNA into doubled strand viral DNA. Integration (4): HIV DNA enters host cell nucleus and

integrates into host cell genome with the help of viral integrase. Replication (5): Integrated HIV DNA

uses host cell machinery to translate viral proteins and transcribe viral RNA. Assembly (6): the

components of HIV including the HIV RNA and proteins move to the cell membrane to form an

immature viral particle. Budding (7): The new immature viral particles are released from cell membrane

and become mature and infectious after viral protease action. 1C. HIV latent infection: In HIV latent

infection, HIV life cycle stops at stage 4 after HIV DNA genome integration. However, under certain

conditions, the integrated HIV genome can be activated and the whole life cycle described in 1B is

accomplished.

Figure 2: CRISPR/Cas9 protein structure and working mechanism

A. CRISP/Cas9 protein domain structure: CRISPR/Cas9 protein structure is illustrated above. The

number of amino acids are labeled above the protein schema, and the domain names are listed below

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the protein schema. HNH nuclease domain is labeled in green, and RuvC nuclease domain consists of

three sub-domains are labeled in red. B. CRISPR/Cas9 working mechanism: In detail, Cas9 has two

nuclease domains (HNH and RuvC) represented by two grey oval shapes in the figure. Each nuclease

domain can cleave a DNA strand directed by a sgRNA (In this figure, a sgRNA is in a black box) which is

complementary to the target DNA sequence. The prerequisite to be a target sequence is the presence of

a NGG (In this figure, it is CGG) sequence downstream of the target site (3’ end). The length of the target

sequence is generally 20 nucleotides (In this figure the target sequence is in green). In cell nucleus, Cas9,

sgRNA and double-strand target DNA form a complex. Each nuclease domain cleave a DNA strand at the

third nucleotide from the NGG (indicated by a thin red line). The double-strand breaks are consequently

repaired mainly by either non-homologous end-joining (NHEJ) or homology directed repair (HDR) when

there is a homologous repair template (not shown). NHEJ is error prone and often results in deletions

and insertions while HDR is a high-fidelity repair according to the homologous repair template. In this

figure, an insertion NHEJ is shown in underlined bold black and a faithful HDR is shown in underlined

bold green.

Figure 3: Application of CRISPR/Cas9 technology to eradicate HIV reservoir cells

The major application of CRISPR/Cas9 technology to eradicate HIV latent reservoir cells (A) is to

eliminate (B) or disrupt the HIV provirus (C) from host reservoir cells genome. In detail, several sgRNAs

which target two or more sites of HIV provirus are applied to HIV reservoir cells with Cas9. This action

results in multiple cuts at the multi-sites of HIV provirus, thus provirus is removed from host cell DNA (B)

or provirus is disrupted (C). The DNA of host HIV reservoir cells is further repaired by DNA non-

homologous end-joining or homology directed repair when the homologous repair template is available

(not shown).

Figure 4. Brain targeting delivery by magnetic or ligand labeled nano particles

16

© 2017 Macmillan Publishers Limited. All rights reserved.

Drug and reagents loaded nano particles are systematically administered and target the CNS cells in

human brain. In magnetic targeting (A), nanoparticles complexes target brain cells and cross the BBB

under the force of magnetic field. In Receptor mediated transcytosis (B), nanoparticle complexes target

brain cells and cross BBB by brain-specific receptor mediated transcytosis. After crossing the BBB, drugs

are released to conduct the therapeutic procedure.

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© 2017 Macmillan Publishers Limited. All rights reserved.
© 2017 Macmillan Publishers Limited. All rights reserved.
© 2017 Macmillan Publishers Limited. All rights reserved.