CRISPR-Cas9 as an advance tool for eliminating HIV/AIDS

Shubham Koirala, alumnus at Kathmandu University, Nepal

HIV/AIDS

Acquired Immunodeficiency Syndrome (AIDS) was first proclaimed when opportunistic infections and rare malignancies were seen among homosexual men in 1981. It is endorsed that AIDS is caused by Human Immunodeficiency Virus 1 (HIV-1) which is also widely known as Lentivirus (belongs to the Retrovirus family). HIV is transmitted through sexual, perinatal, and percutaneous routes, however, 80% of adults attain HIV-1 resulting from exposure at the mucosal surface. This is characterized by the presence of mucosal fluids such as tears, saliva, nasal, cervical, and bronchial mucus, through which the virus may enter, and this is what makes AIDS a Sexually Transmitted Disease (STD). United Nations claimed, as of December 2020 that about 37.6 million people across the globe are living with HIV infection among which 35.9 million were adults and 1.7 million were children and 34.7 million have died from AIDS-related illness since the start of the epidemic.

Molecular basis of HIV infection

HIV-1 has evolved into three distinct groups; N, O, and M. N and O are confined to Cameroon and surrounding countries, whereas the M group is cosmopolitan and its subtypes (A, B, C, D, F, G, H, J, and K) are responsible for about 98% of infection worldwide. HIV-1 virions consist of two identical 9.2 kb single-stranded RNA molecules, however, within the infected host cells, the double-stranded DNA form persists. The DNA genome is flanked at both ends by LTR (Long Terminal Repeat) sequences of which the 5′ LTR region codes for the promoter for transcription of the viral genes. Altogether there are 10 viral proteins which are assisting in viral invasion and replication. Infection of virions is upfront mediated by gp120 (extracellular domain of HIV-1), CD4 (major receptor of the host cell), C-C chemokine receptor type 5 (CCR5), and CXCR4 (co-receptor of the host cell). After binding to co-receptors, the viral core is released into the cytoplasm. Soon after this event, the viral RNA genome is retro-transcribed to the DNA by reverse transcriptase (RT). The pre-integration complex docks to the nuclear membrane and enters the nucleus through the nuclear pore and integrate into host genomic DNA by integrase. The new viral RNA generated by proviral DNA can be used as genomic RNA to make viral proteins. These proteins along with viral RNA migrate towards the cell surface and form immature virus particles followed by their release from cells and yield viral protease which can break down the protein chain to form a mature virus.

Therapies involved in HIV/AIDS

Conventional HIV management includes the administration of Anti-Retroviral Therapy (ART) or Highly Active Anti-Retroviral Therapy (HAART). Only 66.8% of the infected population had access to ART in 2019. Both of these are considered conventional therapy, but due to the lack of advancement in molecular medicine, it has been irreplaceable. Conventional therapies still remain the key therapeutic strategy because they successfully inhibit the HIV replication cycle thereby reducing the viral load and disease progression. Although these therapies have reduced both mortality and morbidity due to HIV-AIDS, they are unable to eliminate the virus completely from the diseased body because HIV remains in the host genome as latent viral form, which upholds the risk of disease recurrence with treatment interruptions. Thus HIV-AIDS is considered a chronic and incurable disease. This secured the need for therapeutic strategies which are not only capable of inhibiting viral replication but also can eliminate latent HIV provirus and reduce the risk of disease recurrence. One of the approaches to achieve this is to adapt therapies that are based on genome editing. In recent days, several studies are being conducted and have been notably interesting.

CRISPR-Cas System

CRISPR is undoubtedly one of the most remarkable discoveries of the era and has the potential to expand the domains of basic and clinical research. The presence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Proteins (Cas) system was first observed in Escherichia coli K12 gram-negative bacteria in the late 1980s. Much later in the mid-2000s, the role of the CRISPR sequence in bacterial defense was comprehended. Viruses or bacteriophages attack bacteria by binding to their surface proteins and inject their DNA through the bacterial cell wall to hijack the cellular machinery of the bacteria. CRISPR tool inside the cell breaks the foreign DNA and integrates sequences into its own bacterial genome. Thus, in the future, when the bacteria are attacked by the same bacteriophage, the unique sequences of the integrated viral DNA help the bacteria to quickly recognize and eliminate the threat, thus, providing adaptive immunity to bacteria and archaea. In a while, the discovery of Cas protein and its affairs in bacteria laid the foundation of the CRISPR-Cas system as a genome-editing tool.

Contrary to existing genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs), the CRISPR-Cas system offers simple, site-specific, and precise genome editing leverage. Genetic editing by the CRISPR-Cas technique relies on the coordinated functioning of Cas protein and chimeric single guide RNA (sgRNA) having a complementary sequence to target DNA.

Molecular basic of CRISPR-Cas system

Out of various systems available, the CRISPR-Cas9 system is highly studied and the crystal structure of Cas9 endonuclease is adequately archived. One of the main components of the CRISPR-Cas System is sgRNA and it is designed in such a way that it has a complementary sequence to target DNA. The main function of sgRNA is to direct Cas endonuclease and other CRISPR components to the target site via complementary base pairing. It finds a specific site present in target DNA known as Proto-spacer Adjacent Motif (PAM). PAM is a special site in target DNA which gives its uniqueness and differentiates target DNA from non-target DNA. The interaction of sgRNA and PAM via complementary base pairing activates the Cas9 endonuclease. Cleavage of target strand is executed by histidine-asparagine-histidine (HNH) domain whereas of the non-target strand is by RuvC domain of Cas9 endonuclease respectively. The cleavage creates a double-stranded DNA break (DSB), which is repaired either by non-homologous end-joining (NHEJ) or homology-directed repair (HDR). If we deliver donor DNA to the cell, which has homology with excised DNA, flanking locus targeted by Cas9, it will be used for repair introducing the desired substitution. However, for the NHEJ, it will randomly introduce insertion or deletion.

CRISPR-Cas System in HIV/AIDS Therapy

Back in 2013, the first approach was made to prevent recurrence of HIV/AIDS by disrupting latent HIV-1 provirus and also by suppressing HIV-1 gene expression in Jurkat cell lines. They targeted NF-KB binding cassettes in the U3 region of Long Terminal Repeats (LTR) and Trans Activating Response (TAR) sequences of the R region of the viral genome, respectively. They successfully eliminated internally integrated viral genes from the infected host cell chromosome. The result of this approach was quite promising which encouraged other researchers to conduct similar studies. However, the use of the CRISPR-Cas9 system was questioned due to its high off-target effects. Eventually, in another study, they used Cas9-sgRNA to target the conserved site in the U3 region of LTR, which resulted in the inactivation of viral gene expression and restricted viral replication with no measurable off-target effects.

Later studies were conducted by targeting multiple sites of the HIV-1 genome which resulted in increased efficiency of disruption of the non-integrated proviral genome. The HIV-1 can escape from the cleavage mediated by a single sgRNA, whereas the multiple sgRNA based CRISPR-Cas9 genome editing approach has higher cleavage efficiency compared to a single sgRNA, which is why multiple sgRNA-mediated editing approach could be a promising HIV/AIDS therapeutic strategy. Thus CRISPR-Cas9 technique with a simple approach, high efficiency, and limited off-target effects can be used to cure HIV/AIDS.

In addition to therapeutic possibility, CRISPR technology can be used to prevent the entry of HIV-1 into the host cells. As specified earlier, the entry of HIV-1 into host cells is mediated by host cell receptors such as CD4, CCR5, and CXCR4. Since the CD4 receptor is also expressed by immune cells on their surface and has a crucial affair in the immune system, the inhibition of CD4 receptors is not desirable. Therefore CCR5 and CXCR4 are considered as likely targets for HIV-1 included gene therapy. Gene therapy using ZNFs by editing CCR5 and CXCR4 has already been successfully used, resulting in a significant decrease of HIV-1 provirus to an undetectable level.

In contrast to ZNF, the CRISPR-Cas9 attempt provides suitable target sites with relatively simple design, high efficacy and can also reduce the cost of therapy involved. In 2013, CRISPR-Cas9 was successfully employed to disrupt CCR5 expression in human embryonic kidney (HEK) by transferring Cas9 and sgRNA. Later CRISPR-Cas9 system was used to target the CCR5 gene in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and achieved long-term CCR5 disruption in-vivo, which resulted in inhibition of HIV-1 infection. Stable suppression of CCR5 has already been observed in secondary repopulating Hematopoietic Cells (HSCs), which gives a reference to develop a potential cure for HIV/AIDS by transplanting CCRS modified HSCs in the future. Similarly, 40% disruption of CXCR4 expression has also been reported in CD4+ T cells with knock-in efficiency of up to 20%. Thus, the CRISPR-Cas9 system has the potential of eliminating the latent HIV-1-viral pool from infected cells and preventing the entry of the virus into healthy non-infected cells, thereby preventing the spread of the HIV-1/AIDS infection.

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12. Image source- Provided to Unsplash by National Cancer Institute (NCI), National Istitute of Health (NIH)

Brief about the author:

Shubham Koirala is biotechnologist by profession. He is excited to push the boundaries of molecular medicine, genetics and virology in his future endeavours.

Other Publications:

“In-Silico development of method for the selection of optimal enzymes using L-asparaginase II against Acute Lymphoblastic Leukemia as an example”. https://doi.org/10.1101/2020.10.13.337097

LinkedIn profile link: https://www.linkedin.com/in/shubham-koirala/

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