Clinical Applications of Gene Editing Technologies: HIV

With the event of antiretroviral therapy, HIV-1 infection is now manageable as a chronic disease. However, HIV remains a worldwide epidemic responsible for considerable mortality and morbidity. Although highly active antiretroviral therapy (HARRT) effectively suppresses viral infection replication and even reduces viral loads in HIV patients, its limitations include patient compliance, side effects of long-term therapy and emergence of drug resistance as well as high cost. 

Moreover, although HARRT extends HIV-1 infected patients’ lives, it doesn’t offer a permanent cure. Thus, there is a requirement to develop simpler countermeasures for HIV infection.

Gene therapy may be an appealing method to derive HIV-resistant cells. Interest during this field began following the demonstration that CCR5 could also be a severe co-receptor for HIV. Genome editing has emerged recently as a more precise thanks to engineer cells applied to HIV/AIDS. 

Clinical Application of Gene Editing Technologies

The clinical application of gene editing techniques towards HIV-1 therapy are as followed:

 1. Zinc Finger Nucleases (ZFN)

Taking advantage of the practical and sequence-specific gene modification capability of ZFNs, the technology has been widely utilized for genome editing in various sorts of cells and living organisms. As for HIV-1 gene therapy by ZFNs, most publications have focused on co-receptor CCR5 or CXCR4 disruption but targeting the HIV-1 genome may be a possible alternative strategy.

• CCR5 Disruption: Theoretically, the cellular receptor or co-receptors involved in HIV-1 entry might be used as targets for inhibiting HIV-1 infection. However, the leading viral receptor CD4 plays a crucial role in the system, making its deletion unacceptable. On the opposite hand, individuals with mutations within the HIV co-receptor CCR5 haven’t any significant adaptive immune deficits.  Hence, CCR5 disruption is taken into account as an attractive therapeutic target to dam HIV-1 entry.
• CXCR4 Disruption: Generally, CCR5 is the predominant co-receptor for HIV entry during initial transmission and through the first infection stage. However, once HIV-1 infection is established, it’s ready to choose CXCR4 as an alternate co-receptor. With the emergence of CXCR4 tropic viruses, CCR5 disruption will not be prepared to protect against the spread of HIV-1. 

For this reason, targeted CXCR4 disruption is additionally being considered as a different strategy for inhibiting HIV-1 infection. To date, various studies have demonstrated that targeting CXCR4 can promote HIV-1 resistance.

• Targeted HIV-1 Proviral DNA Disruption: As described above, disruption of CCR5 and CXCR4 can only stop the spread of the latest virus, which could eventually end in a functional cure. However, this may not be sufficient to eradicate the virus from already infected cells. Moreover, disruption of either gene exposes the likelihood of generating an alternative co-receptor using mutants. Therefore, attempts have also been made to eliminate HIV proviral DNA using ZFN technology.
• ZFN Delivery Strategies for HIV Gene Therapy: Delivery of gene therapy vehicles to specific target cells has been a severe bottleneck for translation into human genome therapy. Gene therapy for HIV infection is primarily focused on CD4+ T cells or CD34+ HSC. 

The problem in targeting the cells in vivo is to switch the cell surface in vitro to be used for reinfusion to infected subjects. With the present state of technology, gene therapy for HIV is usually intended to be used in already infected people.

• Specificity of ZFN Targeted Gene Disruption in HIV-1 Therapy: The elemental safety concern for using ZFN CRISPR (clustered regularly interspaced short palindromic repeats) mediated gene expression for human therapy is the specificity of gene targeting. For instance, if the nuclease action mutates unintended targets important for cellular physiology, the treated cell lines may become dysfunctional or even die. Therefore, ZFNs must be carefully designed to avoid off-target cleavage events as far as possible. 

2. Transcription Activator-like Effectors Nucleases (TALENs) :

Transcription activator-like effectors (TALE) are present DNA binding proteins from plant bacterial pathogen Xanthomonas. TALE proteins contain N and C termini for localization and activation and a central domain for specific DNA binding. 

The invention that TALEs use an easy modular code for DNA recognition has provided an alternate platform for genome editing and gene disruption. 

During a short time after discovery, this system has already been widely used for genome editing in different sorts of cells, model organisms, plants, livestock, and even human cell lines. 

3. HIV Elisa Kit :

The RETRO-TEK HIV-1 p24 Antigen ELISA is an enzyme-linked immunoassay used to detect Human Immunodeficiency Virus Type 1 (HIV-1) p24 antigen in various research specimens, including human sera and plasma as well as cell culture media. It can also be used to monitor the purification and biochemical behaviour of HIV-1.

Furthermore, the assay may augment or supplant polymerase measurements traditionally employed to detect the presence of HIV-1. Such enzymatic measurements aren’t HIV-1 specific. The RETRO-TEK HIV-1 ICx/CRx Kit for immune complex dissociation and confirmation of p24 reactivity enhances the detection of p24 antigen in serum or plasma. 

(Note- The RETRO-TEK HIV-1 p24 Antigen ELISA is supplied for research purposes only.)

4. Engineered CRISPR or crispr-cas9 System :

Bacterial genomes contain loci encoding what’s referred to as clustered regularly interspaced palindromic repeats (CRISPR), interspersed with short intervening spacers. While the repeats are identical, the spacers vary in sequence. The CRISPR or crispr-cas system locus is surrounded by a cohort of CRISPR-associated (Cas) genes. 

The transcribed CRISPR RNA (crRNA) products accompany Cas protein/nuclease and guide the complex to the target DNA that is complementary to the spacer sequence, after which the Cas9 nuclease protein cleaves the DNA to make DSB followed by DNA repair by NHEJ or HR as described for ZFN and TALEN. 

This pathway is usually employed by bacteria to destroy foreign invaders (like plasmids and phages) and evade host cells’ natural immunity to reinforce bacterial virulence. This technique has recently been shown to possess enormous potential for gene editing during hosts, including human cells.

Conclusion

The selected gene-editing method and the off-target effects need careful evaluation at the genome-wide level. The rapid application of high-throughput analysis via deep sequencing methods offers hope that this problem will be soon solved.

In summary, the rapid progress within the development of newer clinical trial application of gene editing technologies offers hope for using these technologies for actual HIV gene therapy within the near future.

To learn more about the clinical application of gene editing technologies: HIV, contact Helvetica Health Care.