CRISPR/Cas9 as an assistant in the fight against HIV

Anna Shmakova, "Biomolecule"

AIDS and HIV infection are still incurable diseases with which 35 million people live in the world. Modern therapy, aimed mainly at inhibiting HIV enzymes, does not affect the provirus in any way – that is, the DNA of the virus embedded in our genome. It is possible to get rid of the virus in our genome thanks to the revolutionary CRISPR/Cas9 system, which will allow cutting out HIV genes.

CRISPR/Cas9

Even 5 years ago, no one could have believed that such a revolutionary approach to the treatment of diseases would appear. The system of "natural" directed editing of the CRISPR/Cas9 genome made a splash in the world of science [1, 2]. This system was discovered almost thirty years ago in bacteria, and then in archaea [3, 4]. It all started with the fact that repeating DNA sections with a length of 20-50 nucleotides were found in the genome of microorganisms, it is unknown why they were needed and separated by unique sequences (spacers) of the same length [5]. In close proximity to these repeats were the genes of various proteins that cut and unravel DNA (cas genes). Genetic analysis has shown that these unique DNA sites are homologous to various DNA sites (protospacers) of bacteriophages and plasmids – enemies of bacteria. Moreover, having the appropriate spacer, the bacterium becomes resistant to the penetration of foreign DNA [3]. Thus, it was shown that the CRISPR/Cas system is a kind of bacterial "immunity" system (Fig. 1).

Figure 1. CRISPR/Cas9 system as one of the variants of bacterial immunity. a – The introduction of foreign DNA into the bacterial cell. b – Acquisition of a spacer. b – Transcription of the CRISPR locus. g – Formation of guideRNA. d – Formation of the active Cas9 + guideRNA complex. e – Binding of foreign DNA. g – Introduction of a double-stranded break in DNA. See abbreviations in the text. Figure from [6].

Cas proteins, which combine with the RNA transcription product of spacers, complementary to the alien gene site, are on guard of immunity. This RNA is a "sketch" of the criminal, with which the policeman is looking for the criminal himself. Having found it, the policeman, that is, the Cas-protein, cuts the intruder [6].

But then in 2012, the bacterial defense system was adopted by scientists, because if you combine Cas9 with a certain RNA, you can cut the necessary gene. The cut gene triggers a repair system in the cell, which literally tries to connect the ends with the ends. At the same time, various mutations of the repaired gene occur – mainly deletions, but there are also different insertions. However, if we provide a matrix for repair, the gene can return to the correct form. So you can make any changes to the genome. This means that in theory we can treat genetic diseases, cancer and more. Today we will talk about how CRISPR/Cas9 will help solve the problem of getting rid of the human immunodeficiency virus (HIV).

HIV

HIV – the causative agent of acquired immunodeficiency syndrome (AIDS) – belongs to the genus of lentiviruses of the retrovirus family, whose genome is represented by two copies of single-stranded RNA, at the two ends of which there are long terminal repeats (or in English long terminal repeat, LTR) [7]. The viral particle itself contains three more enzymes: protease, reverse transcriptase and integrase (Fig. 2). Protease cleaves the products of reading viral genes to form mature proteins.

Figure 2. Structure of the HIV virion. The particle is covered with a lipid bilayer originating from the cell membrane of the host organism, and is dotted with molecules of viral glycoproteins. Drawing from the website agscientific.com .

The life cycle of HIV is quite simple (Fig. 3). Initially, the virus infects the cell, and the CD4 molecule serves as a receptor, and chemokine receptors become coreceptors, therefore, cells of the immune system are affected: T-lymphocytes (T-helper cells), monocytes, macrophages, as well as brain cells (macrophages, microglia, astrocytes), cells of the lymphoid tissue of the gastrointestinal tract and others. Then the single-stranded RNA genome is converted by reverse transcriptase into double-stranded DNA, the so-called proviral DNA. Finally, integrase embeds double-stranded proviral DNA into the host genome. This embedded information is read, and the promoters (that is, the places that attract RNA polymerase for transcription) are long terminal repeats - LTR. New viral particles are collected, infecting new cells. In general, this whole process leads to frequent mutations of the virus, especially the surface glycoproteins are variable, which is also why HIV particles are so elusive for our immunity [8, 9].

Figure 3. HIV life cycle. 1 – mature viral particle; 2 – binding to the cell due to receptors; 3 – fusion of virus and cell membranes; 4 – release of viral RNA; 5 – conversion of RNA into DNA (reverse transcription); 6 – integration into the cell genome; 7 – reading of viral information; 8 – assembly and exit of the viral particle; 9 – a new virus particle. Drawing from the website dentalcare.com .

HIV therapy

Figure 4. Removal of the provirus from the human genome will make it impossible for the virus to reproduce and spread. Figure from [11].

Modern highly active antiretroviral therapy is based on the inhibition of reverse transcriptase, protease, integrase, fusion of the virus with the cell, which significantly complicates the life of the virus. Nevertheless, HIV infection remains an incurable disease at the moment, since such therapy does not affect the DNA of the virus in our genome in any way: in patients receiving antiretroviral therapy, about 106 cells contain provirus. Moreover, patients may have serious side effects or resistance to antiretroviral therapy [10].

Figure 5. The CRISPR/Cas9 system integrated into cells can prevent their infection with the virus and the integration of the virus into the genome. Figure from [11].

Even during therapy, viral genes can be read at some level, which causes various complications in patients with HIV infection, and when therapy is stopped, the virus can "activate". It is proposed to get rid of these pathogenic records in our DNA using the CRISPR/Cas9 system, if it recognizes sections of the viral genome and removes them. This approach can lead to complete elimination of HIV infection (Fig. 4).

The proposed system was tested by Japanese scientists in 2013 on human T-lymphocyte cell culture [8]. They suggested using LTR as a target, and in them there are different sites necessary for binding initiation factors and transcription elongation. Three-fold introduction of the CRISPR/Cas9 construct into cells reduced the reading of the viral genome by almost 3 times (from 97.8% to 35.5%). Further analysis showed that almost all cells contained various mutations in the provirus region.

Since LTRs are terminal repeats, cutting from two ends can theoretically lead to the complete cutting out of the viral piece embedded in the genome. And indeed, almost a third of the cells (31.8%) had this area completely removed after three–fold introduction of the CRISPR/Cas9 system.

What about the mutations of the virus? After all, if the target site changes its structure, the CRISPR/Cas9 system will not be able to find and neutralize it. Realizing this, the scientists chose exactly the LTR site that contains highly conserved DNA sequences that are almost identical in all HIV subspecies.

In 2014, these studies were conducted by American scientists on microglia cells, macrophages and monocytes, confirming the success of such therapy [9]. In addition, scientists have managed to immunize cells from HIV: they ensured that the cells constantly contained the CRISPR/Cas9 system with the corresponding anti-HIV RNA (that is, integrated it into the genome). This did not harm the cells, and after HIV infection, the virus was not inserted into the genome and propagated in them [9, 11, 12]. Who knows, maybe in the future we will all be "vaccinated" against HIV in this way (Fig. 5)?

It's not that simple

The proposed system of gene modification is not as wonderful as it may seem at first glance, no matter how hard I try to convince you of this. There are many pitfalls that require further investigation.

One of the main problems of this method remains its effectiveness. Often, work on the targeted change of one site requires a lot of time and money, but does not lead to a 100% result: not all targets can be found and corrected.

Another problem is the correction of other DNA sections similar to the desired sequence, or so–called off-target effects that can affect the gene, even if we did not want to change it. This problem can be eliminated if you pre-select a sequence that occurs only in the gene that should become the target of Cas9. So, in the already mentioned study, scientists, having selected the correct spacer sequence, complementary to the viral genome, did not find unnecessary mutations in the human genome after using CRISPR/Cas9 [9]. There is another way to increase the specificity: to direct Cas9 not to one site, but to two closely located gene sites, cutting in them not two DNA chains at once, but one in the first target, the second in the second (Fig. 6). Such systems are called nicases. The cell response will be the same as with a double-stranded DNA break, but the specificity is higher, because the policeman now has two sketches of the criminal [12]. It is also proposed to configure this system so that it can be "turned on" and "turned off" after use, so that it does not work constantly in the cage.

Figure 6. Increasing the specificity of gene cutting using nicases introducing single-stranded breaks. Figure from [6].

And finally, the problem of CRISPR/Cas9 system delivery. We have already learned how to deliver it to individual cells, but how to act effectively at the level of the human body, where there are about 10 ⁶ infected cells? There are different approaches to such gene therapy: delivery using viral vectors (viruses that "infect" us with good genes), the use of nanoparticles [13], the collection of bone marrow stem cells, the introduction of CRISPR/Cas9 into them and subsequent reverse transplantation [9]. When developing such therapy, it should also be taken into account that brain cells also serve as a reservoir of the virus, where it is not so easy for large structures to penetrate [11].

Thus, rapidly developing gene therapy using CRISPR/Cas9 is the key to getting rid of HIV infection and associated diseases and complications. However, one should not expect that such a medicine will appear on the shelves in a pharmacy today or tomorrow, because there is still a lot to think about in order to develop a truly effective and safe treatment.

Literature

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Portal "Eternal youth" http://vechnayamolodost.ru  19.10.2016