Application of Gene Editing Technologies in HIV Infection and

Prevention

Zhihang Li

and Haotian Luo

Shanghai Institute of Technology University, Shanghai, Shanghai, China

Maple Leaf International School‑Chongqing, Chongqing, Chongqing, China

Keywords: Gene Editing, HIV Prevention, CCR5.

Abstract: Human immunodeficiency virus (HIV) causes AIDS by interacting with host receptors like CD4, CCR5, and

CXCR4. While antiretroviral therapies suppress viral replication, they don't provide a cure, and drug

resistance is a persistent problem. Gene editing technologies, such as CRISPR-Cas9, TALEN, and artificial

miRNAs, offer promising solutions for HIV prevention and treatment. By mimicking the CCR5Δ32 mutation,

these technologies can block HIV entry into host cells. This review explores HIV infection mechanisms, the

role of CCR5 in viral entry, and gene editing strategies to modify CCR5. It also examines animal models for

cross-species infection and highlights successful HIV cure cases (the Berlin and London patients). The review

provides insights into future gene editing strategies for HIV cure research, focusing on overcoming challenges

and advancing treatment possibilities.

1 INTRODUCTION

The human immunodeficiency virus (HIV) is a

lentivirus that causes AIDS by interacting with

different types of cells in the body and evading the

host's immune response to it. HIV is mainly

transmitted through blood and reproductive fluids and

can be transmitted from an infected mother to a

newborn (Levy et al., 1993). The process of infection

involves not only the interaction of HIV with CD4

molecules on the cell surface, but also the binding to

other cell receptors. Subsequently, the virus fuses

with the cell and enters the host cell, where it begins

its replication process. After HIV infection, different

intracellular mechanisms determine whether the virus

triggers productive or latent infection, especially in

CD4+ T cells, where HIV replication can lead to

syncytial formation and cell death; Other immune

cells, such as macrophages, may develop persistent

infections, forming a reservoir of the virus.(Moir et

al., 2011)Although antiretroviral therapies, such as

CCR5 receptor antagonists like maraviroc,

effectively inhibit HIV replication and delay disease

progression, these treatments do not cure the

infection, and drug resistance remains a significant

challenge.

In recent years, the rapid development of gene editing

technologies such as CRISPR-Cas9, TALEN, and

artificial miRNA, has provided new possibilities for

the prevention and treatment of HIV. CRISPR-Cas9

technology involves designing a specific guide RNA,

which directs the Cas9 protein to the target DNA

within the cell. The Cas9 protein, guided by the RNA,

then precisely cleaves the DNA, enabling targeted

gene modification. TALEN technology consists of

two parts: one is the TALE protein, which specifically

recognizes and binds to a specific sequence of DNA;

The other part is the FokI nuclease, which is able to

cleave DNA under the guidance of the TALE protein.

Artificial miRNAs specifically inhibit CCR5

expression through RNA interference mechanisms,

thereby preventing HIV invasion (Kennedy et al.,

2017). The CCR5 receptor plays a crucial role in the

infection process of HIV, which enters the host cell

by binding to the CCR5 receptor. Therefore,

modifying the CCR5 gene to block This pathway is

an effective strategy to prevent HIV infection.

The Berlin and London patients are two of the

world's most famous cases of being cured of AIDS

through mutations in the CCR5 gene. In 2007, the

Berlin patient underwent a bone marrow trans-plant

with the CCR5Δ32/Δ32 mutation for leukemia, and

the HIV virus was completely eliminated after the

operation, becoming the first person in the world to

340

Li, Z. and Luo, H.

Application of Gene Editing Technologies in HIV Infection and Prevention.

DOI: 10.5220/0014488200004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 340-344

ISBN: 978-989-758-789-4

be cured of AIDS. The London patient received a

similar stem cell transplant for Hodgkin lymphoma in

2016 and became the second patient to be cured after

the virus was undetected for more than 3 years after

the drug was stopped. These two successful cases

demonstrate the great potential of CCR5 genetic

modification in blocking HIV infection (Shen et al.,

2022). With the development of gene editing

technology, the modification of CCR5 by means of

CRISPR-Cas9 and TALEN has become a research

hotspot. The purpose of this review is to deeply

explore the application of gene editing technology in

the prevention of HIV infection, especially the

mechanism of blocking the entry of HIV virus into

host cells by modifying the CCR5 receptor, and to

discuss its prospects and challenges in practical

application, so as to pro-vide a theoretical basis for

the development of future HIV prevention strategies.

2 MECHANISM OF HIV

INFECTION AND THE ROLE

OF CCR5 RECEPTORS

Human immunodeficiency virus (HIV) infection has

a unique course characteristic. HIV is mainly

transmitted into the human body through blood,

semen, vaginal secretions, and mother-to-child

transmission. The virus first binds to the CD4

receptor on the surface of CD4+ T cells and then

enters the cell via the CCR5 receptor or CXCR4

receptor. In the early stages of infection, the virus

spreads less efficiently, but in the acute phase, strong

replication occurs and spreads to lymphoid tissues.

This is followed by a prolonged chronic phase, which

is usually asymptomatic but accompanied by

sustained immune activation and viral replication. In

advanced stages, CD4+ T cells are significantly

depleted, eventually leading to acquired immuno-

deficiency syndrome.

The CCR5 receptor plays a crucial role in the

invasion of HIV, especially in the acute phase, when

HIV enters host cells mainly through CCR5. CCR5 is

an important chemokine receptor in the immune

system, which is mainly expressed in CD4+ T cells,

macrophages and other immune cells. Studies have

found that the deletion or mutation of CCR5 receptor

(such as CCR5Δ32 mutation) can significantly

improve an individual's resistance to HIV, block the

binding of HIV to CCR5, and prevent the virus from

entering cells, thereby achieving natural immune

protection. The CCR5Δ32 mutation causes a loss of

function in the CCR5 receptor, and individuals with

this mutation are generally less susceptible to HIV

infection. Studies have shown that this mutation can

effectively block the binding of HIV to immune cells

and is an effective natural barrier against HIV, so it

has great potential to be applied to mimic this

mutation to resist HIV infection through gene editing

(Khalili et al., 2017).

3 CRISPR-CAS9 TECHNOLOGY

In nature, bacteria will be attacked by various viruses.

Once they survive the attack of the virus, they will

record some of the genetic characteristics of the virus

and carve it into their own DNA database. If they are

attacked by the same virus next time, a large amount

of RNA will be transcribed from the virus database.

These RNAs contain the genetic characteristics of the

virus, which are called guide RNA. Cells also make a

protein called CAS. CAS proteins bind to guide RNA

to find viruses with this gene signature, and

accurately remove the virus's genes to achieve

immunity to this virus. Based on the discovery of the

bacterial immune system, CRISPR-Cas9 technology

artificially designs a piece of guide RNA according to

the DNA edited needs, and then introduces the DNA

of this piece of guide RNA and CAS protein into the

cells. This will produce a lot of CAS proteins and use

guide RNA to accurately cleave the DNA that needs

to be edited. When DNA breaks, cells look for

fragments that are the same as the sequence at the

fracture for recombination repair. At this stage,

multiple copies of the same DNA fragments at the

artificially designed break site are introduced,

allowing the cells to use them as a template for

recombination and repair, thereby achieving precise

gene editing (Jinek et al., 2012).

By manually designing a guide RNA for the

CCR5 receptor protein, the DNA of this RNA and

CAS protein is introduced into the cell. Then, the

CAS protein will accurately cleave the CCR5

receptor protein under the guidance of guide RNA. In

this way, the HIV virus cannot recognize helper T

cells, thereby achieving immunity to HIV. In

Christian L. Boutwell's study, four best gRNAs were

tested and found that they were highly editable in T

cells, ranging from 52% - 70%, and both effectively

reduced CCR5 expression on the surface of CD4+

and CD8+ T cells. The "dual guide" method used to

combine two of these gRNAs can effectively edit the

CCR5 gene and reduce CCR5 expression. The

infection rate of all CCR5-edited CD4+ T cells was

Application of Gene Editing Technologies in HIV Infection and Prevention

341

significantly reduced, indicating that CCR5-edited

can effectively confer the CD4+ T cells against HIV.

Later, mice were used for experiments, and in mice

transplanted with CCR5-edited HSPC, CCR5-

expressed CD4+ T cells were significantly reduced.

The researchers infected mice with high doses of

HIV, and the results of CCR5-edited mice showed

complete resistance to HIV infection, which strongly

demonstrated that high-frequency CCR5 editing can

effectively confer protective effects on HIV

(Claiborne et al., 2025).

4 TALEN TECHNOLOGY

TALEN technology refers to the use of two parts of

DNA recognition domain and endonuclease to form a

protein as a gene knockout tool. Scientists have

discovered a bacterial protein (TALE), whose

dichotomous amino acid corresponds one by one to

the four bases. NI recognizes A, NG recognizes T,

HD recognizes C, NN recognizes G. Therefore,

TALE can be used as a DNA recognition tool to

cleave DNA in site-pointed with FokI dimer.(Boch et

al., 2014 )A study designed two pairs of TALEs for

the CCR5 gene, targeting specific regions of the DNA

double-stranded, ensuring that the recognition

sequence length is 18-20 bp to enhance specificity. It

is then combined with FokI to form a protein to

accurately knock out CCR5 receptor DNA. The

produced protein was injected into CD4+ T cells and

hematopoietic stem cells using lentiviral vectors, and

the expression level of CCR5 receptors was

confirmed through in vitro experiments and the

knockout rate was confirmed. Finally, the HIV-1

virus (R5 tropic strain) was introduced into the cells

to test their resistance to the virus. The results show

that the CCR5 receptor knockout rate exceeds 60%, it

is very resistant to HIV-1 virus, the amount of virus

is reduced by 90%, and can still maintain stable

effects under long-term exposure (Shi et al., 2017).

5 ARTIFICIAL mirNA

TECHNOLOGY

Artificial miRNA technology targets and silences the

expression of specific genes by introducing specific

miRNA sequences. Inside the nucleus, genomic DNA

is transcribed into primiRNA and then processed by

Drosha enzymes into pre-miRNA, with a length of

about 70 - 100 nucleotides. Then, the Dicer enzyme

in the cytoplasm cleaves pre-miRNA into double-

stranded miR-NA. One strand in a double-stranded

miRNA binds to the AGO protein, constituting an

RNA-induced silencing complex (RISC) and forming

a mature RISC-miRNA complex. RISC-miRNA

complex recognizes targets through base

complementary pairing of miRNA with target

mRNA. After binding to the target mRNA, the RISC-

miRNA complex achieves gene silencing in two

ways. The first is to inhibit translation, which inhibits

protein synthesis by hindering the binding of

ribosomes to mRNA or affecting the formation of

translation initiation complexes. The second method

promotes the degradation of mRNA: attracts

nucleases to cleave mRNA, causing it to degrade,

resulting in a decrease in the expression level of the

target gene (Duchaine et al., 2019). One study used

Western blotting to analyze the expression levels of

CCR5 protein by transfecting miRNA-103 to CD4+

T lymphocytes, followed by quantitative real-time

polymerase chain reaction to detect expression levels

of miRNA-103 and CCR5 mRNA, and flow

cytometry was used to detect CCR5 expression and

HIV-1 infection on the cell surface. Later, through

mouse experiments, when miRNA-103 expression

was increased, the expression of CCR5 protein was

significantly reduced, and when miRNA-103

expression was inhibited, the CCR5 protein would

rise. This demonstrates that miRNA-103 can regulate

the expression of CCR5 protein. When the CCR5

protein is inhibited, the infection of HIV-1 to cells is

significantly reduced (Bellini et al., 2022).

6 RESEARCH PROGRESS OF

CCR5 GENE EDITING IN HIV

PREVENTION

6.1 Establishment of Animal Models of

HIV Infection

Establishing cross-species animal models for HIV

infection is essential for advancing our understanding

of HIV/AIDS pathogenesis. However, HIV-1 is

species-specific and only infects humans and a few

non-human primates, making it challenging to study

in traditional animal models such as mice. A recent

study successfully created a mouse model capable of

being infected with HIV-1. By introducing the CD4,

CCR5, and CyclinT1 genes into the mouse leukemia

cell line L1210 using lentiviral vectors, the

researchers enabled these cells to express the

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necessary receptors and co-receptors for HIV

infection. Fluorescence analysis and sequencing

confirmed the significant expression of CD4, CCR5,

and CyclinT1 proteins in the transgenic cells, and

HIV-1 RNA was detected in the culture medium,

indicating successful virus entry and replication. This

model provides a critical platform for studying HIV-

1 cross-species infection and offers new directions for

HIV vaccine development, antiviral drug screening,

and further exploration of HIV/AIDS pathogenesis

(Karuppusamy et al., 2021).

6.2 Cases of AIDS Cure

The "Berlin Patient," Timothy Ray Brown, became

the first person in the world to be cured of AIDS

following a bone marrow transplant in 2007. Brown,

who was also battling leukemia, received a transplant

from a donor with the CCR5Δ32 mutation, which

naturally blocks HIV entry into cells. After the

procedure, not only was Brown’s leukemia

successfully treated, but HIV was undetectable in his

body, effectively achieving a "double cure."

Similarly, the "London Patient," Adam Castillejo,

underwent a hematopoietic stem cell transplant for

Hodgkin's lymphoma in 2016, receiving cells with the

CCR5Δ32 mutation. After discontinuing

antiretroviral therapy, HIV remained undetectable in

his body for several years, with no recurrence of the

infection.

These two groundbreaking cases highlight the

potential of CCR5 gene modification for both

controlling and potentially curing HIV. However, the

procedures involved—particularly bone marrow

transplants—are highly complex, risky, and prone to

serious complications. Moreover, finding matching

donors is exceedingly difficult, limiting the

widespread applicability of this treatment. While

these cases offer hope and insight for CCR5 gene

editing in HIV treatment, they remain exceptional

cases and do not yet represent a practical, widely

accessible solution. Nonetheless, they provide

invaluable direction for ongoing research into CCR5

gene editing and its potential to prevent or cure HIV

infection.

7 CONCLUSION

The HIV infection mechanism highlights the crucial

role of CCR5 receptors in the virus's ability to enter

host cells, making them a key target for potential HIV

prevention and treatment strate-gies. Recent

advancements in gene editing technologies, such as

TALEN, miRNA, and CRISPR/Cas9, have

demonstrated the potential to modify or disrupt

CCR5, effectively prevent-ing HIV from entering

CD4+ T cells. These innovations not only offer a

promising approach to blocking HIV infection but

also open new possibilities for a functional cure by

mimicking the natural CCR5Δ32 mutation that

provides resistance to HIV. However, despite these

promising developments, several challenges remain

in the application of gene editing for HIV prevention

and treatment. Issues related to the accuracy of gene

editing, off-target effects, and the safety of these

technologies must be addressed before they can be

widely adopted in clinical practice. Additionally,

ethical concerns surrounding gene editing,

particularly in human germline modifications, need to

be carefully considered and regulated. Ongoing

research and clinical trials are essential to refine these

technologies, ensure safety, and overcome ethical and

legal barriers, ultimately paving the way for gene

editing to become a transformative tool in HIV

prevention and potential cure strategies.

AUTHORS CONTRIBUTION

All the authors contributed equally and their names

were listed in alphabetical order.

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