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Gene Editing Technologies: HIV

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Gene Editing Technologies Show Promise in HIV Research



The development of CRISPR/Cas systems for editing genes is revolutionizing genetic engineering.1, 2 Biological pathways associated with disease processes such as HIV infection are of particular interest.3 HIV causes severe damage to the immune system by infecting and destroying cells of the immune system, particularly T cells which are important for mammalian adaptive immune responses. CRISPR/Cas gene editing models have been developed by different research laboratories in attempts to curtail HIV infection.

CRISPR/Cas was initially discovered as a key component of prokaryotic adaptive immune systems.

CRISPR stands for clustered regularly interspaced short palindromic repeats, which are segments of prokaryotic DNA containing short repetitions of base DNA sequences. The repetitions are separated by short spacer foreign DNA, originating from previous exposures of the prokaryotes (bacteria and archaea) to plasmids or bacteriophage viruses.

CRISPR functions in coordination with CRISPR associated (CAS) genes, enabling prokaryotic adaptive immune systems to recognize, respond to, and eliminate foreign DNA. CRISPR/Cas gene editing is referred to as a molecular scissors because it essentially cuts the foreign DNA (plasmid and bacteriophage) out of the prokaryotic genome. Analogies of CRISPR/Cas gene editing in prokaryotes have been made to RNA interference or gene silencing in eukaryotic organisms.

Prokaryotic CRISPR/Cas mechanisms are being exploited to develop a rich array of editing tools such as CRISPR/Cas9 for mammalian cells. CRISPR/Cas9 has two main components: Cas9 and a RNA guide, which targets Cas9 to a specific stretch of DNA by complementary nucleotide binding. Cas9 is an endonuclease that cleaves double-stranded DNA in a sequence-specific way through association with guide RNA. The guide RNA contains nucleotides that pair with the target DNA.

RNA guides are genetically engineered to pair with gene targets of choice and delivered to cells along with Cas9. When the RNA guides Cas9 to the targeted genome sequence, it allows Cas9 to cut both strands of DNA at the selected, targeted point in the genome. DNA can then be modified, inserted, or removed at the Cas9 cut sites.

Researchers have designed RNA guides to direct Cas9 to cleave different regions of HIV viral DNA containing essential genes or the long terminal repeat.3 CRISPR/Cas9 gene editing resulted in significant suppression of HIV viral production and infection in various research cell models, including human pluripotent stem cells, primary CD4+ T cells, and CD4+ T cell lines.

Theoretically, the elimination of HIV viral DNA or genes essential for viral expression in infected cells should be able to inactivate or get rid of HIV infection.



Unfortunately, researchers also showed cells developed CRISPR/Cas9 resistance mutations in some experiments. These mutations clustered at the viral site when the Cas9 was directed to cleave. Cas9 was unable to cleave the targeted site and hence the CRISPR/CAS9 attacks were ineffective.3 HIV, like other viruses, was already known to have a well-developed ability to evolve resistance to other attacking mechanisms such as the human immune system and antiviral drugs.

In another approach for combating HIV-1 infection, CRISPR/Cas9 has been used to ablate chemokine receptor type 5 (CCR5) expression. 4 CCR5 is a co-receptor and required for certain HIV types to gain entry into T-cells and cause HIV infection. Researchers disrupted the CCR5 gene in CD34 positive hematopoietic stem and progenitor cells with CCR5-targeting CRISPR/CAS9 vectors. Researchers showed that CCR-5 mutated clones retained their ability to differentiate into multiple hematopoietic lineages and that few off-target mutations were introduced. This is important because safety is high among the concerns of gene editing technology. Hence, it is essential to demonstrate that edited cells can still function and unintended, possibly harmful mutations are avoided. Both successes and recognition of limitations of CRISPR/CAS systems are fueling strategies for optimization.

Importantly, the strategy to mutate CCR5 has been successfully tested in initial clinical medical research trials using zinc-finger nucleases (ZFNs). ZFNs, like CRISP/Cas, are also promising gene editing molecular scissor tools for curtailing HIV expression in cells. ZFNs are generated by fusing a DNA-cleavage nuclease domain to a Zinc-finger DNA binding domain. Like the RNA guides used in CRISPR/Cas technology, Zinc-finger domains can be engineered to target specific DNA. The nuclease domain containing the Fok1 restriction enzyme, is the molecular scissors component and catalytically cleaves targeted DNA. SB-728-T, also known as CCR5-ZFN, is an investigational gene therapy ZFN agent (clinicaltrials.gov: NCT01543152, NCT01252641, and NCT01044654).

The mechanism of CCR5-ZFN is based on genetically engineered modifications of CCR5 which make CCR5 non-functional and cells more resistant to HIV infection. CCR5-ZFN agents are autologous T-cells such as CD4+ T-cells, obtained from a given individual, which are genetically modified at the CCR5 gene by zinc finger nucleases and reintroduced into the host organism. The modified CCR5 locus potentially eliminates one venue (CCR5 receptor) for HIV to gain entry into and infect T cells.4 CCR5-ZFN/SB-728-T is being used for research to determine if CCR5 genetically modified cells are resistant to HIV infection.

Researchers are also eager to determine if CCR5-ZFN/SB-728-T can replicate itself into additional resistant cells and can restore immune cell function in infected hosts by preventing HIV entry into cells.



MyBioSource offers the following products for Gene Editing Research Uses:

• Human Chemokine Receptor Type 5 (CCR5), ELISA Kit (Catalog #MBS2020083)
• CRISPR/Cas9, Monoclonal Antibody (Catalog #MBS375383)
• S. Pyogenes CRISPR-Associated Protein-9 Nuclease, Recombinant Protein (Catalog #MBS140642)
• HIV (Human Immunodeficiency Virus) ELISA Kit (Catalog #MBS766122)
• HIV-2 gp39, Monoclonal Antibody (Catalog #MBS430025)
• HIV Gp41/gg36, Recombinant Protein (Catalog #MBS319756)
• HIV-1 p24, Monoclonal Antibody (Catalog #MBS8241470)
• HIV-1 Tat interactive protein 2 Monoclonal Antibody ELISA Kit (Catalog #MBS732587)
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References
1. Mojica FJ M, Rodriguez-Valera, F. The discovery of CRISPR in archaea and bacteria. 2016. FEBS J, 283: 3162-3169. doi:10.1111/febs.
2. Tschaharganeh DF, Lowe SW, Garipp RJ, Livshits G. Using CRISPR/Cas to study gene function and model disease in vivo. 2016. FEBS J, 283: 3194-3203. doi:10.1111/febs.13750
3. Liang C, Wainberg MA, Das, AT, Berkhout B. 2016. CRISPR/Cas9: a double-edged sword when used to combat HIV infection. Retrovirology, 13:37. doi:10.1186/s12977-016-0270-0
4. Savic N, Schwank G. 2016 Advances in therapeutic CRISPR/Cas9 genome editing. Translational Res 168:15-21Khan doi: http://dx.doi.org/10.1016/j.trsl.2015.09.008
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