GENETICSxHIV

Over the past years, there had been a great number of diseases. Some of these diseases were not yet even identified before. But with the help of science and technology, more conditions are being discovered including the mechanisms on how they work. One significant virus that was discovered to exist in the late 1970s was a virus causing AIDS. It was not until 1981 that the scientists identified what kind of virus was causing it. Eventually the doctors found out that there were some people who were resistant to the said virus. What might be causing this?

DNA Double Helix Turns FiftyDifferent people have different kinds of genes. Each one of us is unique in one way or another. Genes are the ones who are responsible in expressing the trait, both genotypically and phenotypically, that each one of us carries. Depending on what comprises these genes, a variety of characteristics are being expressed by different individuals. In the case of HIV, the answer to the question why there were people who seem to be resistant to HIV infection lies in the mechanism on how HIV works within our bodies, and how they interact with our cells to be specific.

HIV virus shutterstock_236439394All viruses including HIV cannot make copies of themselves without the help of other cells. The virus first needs to enter a cell. But how does this work? There are several proteins that aid them in entering the cells. In the case of HIV, a CD4 receptor is needed by the virus to attach to CD4 cells. It is well established that these CD4 cells play a very important role in our immune system, combating different foreign bodies. When the virus has already attached to the cell, it then has the capacity to destroy it. The destruction of the CD4 cells causes the immune system to bag down eventually. If CD4 cells are not enough, the help of another protein named CCR5 is needed by the virus for it to enter the cells and make copies for itself.

Viruses are known to exploit host proteins for entry, replication, and transmission. This ability is mainly due to their small size and limited number of encoded proteins. These proteins are known as the host dependency factors and they are used to identify therapeutic agents in genetic interventions. If they are really not necessary or required for cellular viability, then they could be ideal targets for therapeutic interventions. Therapeutic targeting of the host rather than the virus has a much higher resistance to drugs. CCR5 is a receptor for the HIV infection of CD4+ T cells and macrophages. This discovery led to the development of small molecule inhibitors of CCR5, which is very essential in therapeutic interventions. The said host dependent factors present as attractive targets for curative gene therapy. Inactivation of several factors could lead to certain discoveries including treatment and cure. For instance, during the only recorded case of HIV cure, the CCR5 delta 32 allele was inactivated. This happened during a hematopoietic stem cell transplant from a donor who was homozygous for the allele. Unfortunately, individuals who had this allele inactivated presented to have increased susceptibility to viral infections, diseases, and cancer.

During the previous years, scientists had used the RNA-i based screening. However, this test was found out to present high false positive rate or low reproducibility results. Even though there were improvements in the mentioned screening test, issues on sensitivity and specificity still remained. Because of this, the study that this blog will be presenting in the following paragraphs used a different kind of screening test, the CRISPR Test. This is a useful tool for genetic screening experiments because of the relative ease of designing gRNAs and the ability of the Cas9 to modify virtually any genetic locus. This test is also responsible for finding the genes required for cell viability, drug sensitivity and resistance. In this study, the researchers used the CRISPR-Cas9 Based Screening because it uses lentiviral single guided RNA (sgRNA) libraries that enable pooled loss of function screens with greater sensitivity and specificity than RNA-i based screens. The study aimed to (1) identify host factors in a physiologically relevant cell system using a genome wide CRISPR based system and (2) identify 5 factors that are required for HIV infection.

The researchers were able to identify 5 host dependency factors necessary for HIV infection. These factors include the two most common receptors for HIV, which are the CD4 and CCR5 co-receptors.  While the less commonly known factors are TPST2, SLC35B2, and ALCAM which were not identified from commonly used screens. Moreover, researchers found out that the loss of the aforementioned factors could not damage any cell function which suggests that these factors are non-essential host protein to which HIV relies for successful replication. Such findings could lead to possible potential therapeutic interventions.

From the HDF’s identified, different mechanisms were also discovered. One of which is co-delivery of TPST2, one of the HDF’s, to mediate high levels of sulfation and for effective neutralization. Basically, sulfation pathway is important for effective HIV infection, thus, inhibiting TPST2 would partially disrupt HIV infection.  Other mechanism includes that of ALCAM factor which is a receptor of the T cell involved in the adhesion of cells. This is particularly significant in HIV infection since HIV is known to spread more efficiently by direct cell to cell contact than cell-free transmission. Thus, if adhesion or aggregation of the T cells is maneuvered by ALCAM, it could halt the spread of the said infection in vivo.

Globally, HIV patients make use of antiretroviral therapies as conventional treatment, however, these treatments allow the spread of drug-resistant strains causing an increased threat to the human population. Nevertheless, the researchers believe that HIV therapies that target host genes through different HDFs necessary for HIV replication may increase the barrier to drug resistance, allowing to pave way for another curative strategies.

CRISPR-Cas9 is among one of the approaches used by the researcher in the study. What is this approach all about? CRISPR-Cas 9 is one of a kind technology that enables geneticists and researchers to correct parts of the genome and to introduce changes to one or more genes in the genome of a cell interest. This is done by removing, adding, or altering sections of DNA sequence. CRISPR-Cas9 is currently the simplest, most precise, and very useful method of genetic manipulation in the science world as of today. How does it work? The CRISPR-Cas9 system is made up of two key molecules namely Cas9 and guide RNA (gRNA) that introduce a mutation into the DNA. Cas9 is an enzyme that acts as a pair of molecular scissors for cutting the two strands of DNA at a specified location in the genome to add or remove bits of DNA, Guide RNA is a piece of RNA consists of a small piece of pre-designed RNA sequence located within a longer RNA framework. gRNA also is designed to find and bind to a specific sequence in the DNA.

Are there other techniques for altering genes? For several years, scientists learned genetics and gene function by studying the effects of changes or mutation in the DNA. They also used chemical or radiation to cause mutations. However, they had no way of controlling where in the mutation of genome it would occur. For a long time, scientists have been using gene targeting to introduce changes in specific places of genome by removing or adding either the whole genes or single bases. Gene targeting has been very valuable for studying genes and genetics, nevertheless, it is lengthy to create a mutation and at the same time is very expensive. 2014-06-27-top-7-alzheimers-myths-debunked-genesNowadays, several gene editing technologies have been developed in order to improve gene targeting methods, which includes CRISP-Cas systems, transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs). Among those, the CRISPR Cas9 system currently stands out
the fastest, cheapest, and most reliable system for editing genes.

CRISPR-Cas 9 has a lot of potential for treating a range of medical conditions that have a genetic component, such as cancer, hepatitis B, and high cholesterol. Though, there have been a lot of interest and debate about the potential to edit reproductive cells, since any changes in cells that will be passed on from generation to generation will  always have an important ethical implications. It is illegal to carry out gene editing in reproductive cells. On the brighter side, the use of CRISPR-Cas9 and other gene editing technologies in somatic cells is uncontroversial. Indeed, they have already been used to treat human disease on a small number of exceptional life-threatening cases.

To identify host genes that are important in facilitating HIV infection, they engineered a physiologically relevant CD4+ T cell line model suitable for pooled CRISPR-based screening, named ‘GXRCas9. This screening uses flow cytometry to monitor the state of cellular infection at the single cell level. Using this device, they screened CCR5-tropic HIV 1 straing JR-CSF and then performed a pooled genome-wide screening using lentiviral library. They used this particular virus strain since almost all known transmitted founder strains of HIV-1 which established de novo infection in naïve hosts are CCR5-tropic20. 200 million GXRCas9 cells were infected by the library and puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger that inhibits protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation (ThermoFisher Scientific, 2017). After a week, they spin-infected JR-CSF cells and were able to detect the GFP+ population. After two additional weeks, they re-infected the cells and cultured them for an additional 10 days but found no change in viability with the flow cytometry results suggesting that the cells harboured genetic knockouts that made them resistant to HIV infection.

To investigate the specific  properties of ALCAM that may be used in therapeutic interventions, GXRCas9 cells which were previously cultured were converted, targeting the ALCAM HDF; isolating the cells without ALCAM(called ALCAM-null cells) by Fluorescence activated cell-sorting (FACS) . upon reviewing the ALCAM-null effects, it was discovered that ALCAM loss did not affect the cell production and multiplication. Nor did loss of ALCOM show any protection against the JR-CSF stain of virus that was being tested. However, one distinct observation made was about the formation of aggregates between wild-type GXRCas9 cells under the standard culture condition whereas those genetically altered GXRCas9 (ALCAM-null) grew as single cells. The re-expression of ALCAM on the other hand rescued the aggregation phenotype of the cell confirming the precise cellular function of ALCAM which is a cell adhesion molecule on activated T –cells, monocytes, and dendritic cells.

The experimenters also wanted to understand how the loss of TPST2 and SLC35B2 confers protection against HIV infection and found out that under normal culture conditions, TPST2-null and SLC35B2-null cells were viable and proliferated at rates comparable to those of wild-type cells and of knockout cells that were complemented with an sgRNA-resistant cDNA. They also found that loss of either TPST2 or SLC35B2 protected cells from viral entry, and that susceptibility was restored when the inactivated gene was added back.

To extend the physiological relevance, they infected these cell lines with Rejo.c, a CCR5-tropic transmitted founder HIV-1 strain21, and obtained similar results with the cell proliferation assays. Re-expression of an sgRNA-resistant cDNA encoding the missing gene completely ablated this resistance, whereas no changes in HIV susceptibility were seen after transduction with an irrelevant control gene, RAP2A. Confocal microscopy images of this test reveal that TPST2-null and SLC35B2-null cells appeared healthy after the HIV challenge, whereas cells that were transduced with a nontargeting sgRNA had a grossly apoptotic appearance.

To determine the mechanisms by which TPST2 and SLC35B2 facilitate viral entry they tested the importance of cellular sulfation for HIV entry by culturing GXRCas9 cells in medium that was depleted of sulfates and in the presence of sodium chlorate, an inhibitor of sulfation. Using the β-lactamase-based viral fusion assay, they found that sulfate-depleted cells are protected from viral fusion relative to cells cultured under standard conditions.

To investigate the possibility that loss of SLC35B2 protects against HIV infection by depriving TPST2 of PAPS, they assessed CCR5 surface expression by flow cytometry using sulfation-sensitive and sulfation-insensitive CCR5-specific antibodies. They found that nearly all surface CCR5 was sulfated in wild-type GXRCas9 cells, while none was sulfated in TPST2-null and SLC35B2-null cells. The total levels of CCR5 on the surface of these cells were unchanged, and add-back of the relevant gene rescued CCR5 sulfation.

Indeed, our technology is improving right now and computers seem to take over everything:  from hiring, to shopping, and the stock market, that billions of people are connected to a web. Before, this would seem absurd but it all happened. Science fiction became our reality and we don’t even think about it. Today, we’re on the point of genetic engineering, which includes different methods including this so-called CRISPR. This recent breakthrough will truly change how we will live and what we will perceive as normal forever.

When scientists figured out that the CRISPRR system were programmable, it gave interest to the whole world of genetics.  Since, you can just give it a copy of DNA you want to modify and put the system into a living cell. If the old techniques of genetic manipulation were like a map, CRISPR is like a GPS system. Also, CRISPR offers the ability to edit live cells, to switch genes on and off, and target and study particular DNA sequences, including those with HIV, because it works on different types of organisms such as microorganisms and humans. Scientists use CRISPR to cut the HIV virus out of the living cells from the patients in the lab, proving that it was possible.

Hopefully in a few decades, a CRISPR therapy might cure HIV and other retroviruses. Even though CRISPR is powerful – it is not that perfect yet. Wrong edits might still happen in the DNA which may be unnoticed.  That is why working on accuracy and monitoring methods is always a major concern. This possible positive future of curing infectious disease can also have darker visions too. Technology certainly makes us worry, but we have a lot to achieve, and genetic engineering, including CRISPR, is just a step on the evolution of the future we are always thinking about. Our world is always full of opportunities and challenges. We might end HIV.

References:

https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

https://www.thermofisher.com/ph/en/home/life-science/cell-culture/transfection/selection/puromycin.html

Advertisements