To what extent can recent discoveries in the field of genetic and epigenetic editing impact the treatment of viral and bacterial infections?

By Rodion Senko

Tools such as CRISPR-cas9 and CRISPR-cas13 are what made the recent advances in genetic and epigenetic editing possible and have led to many applications of genetic editing in medicine, formally called gene therapy. These discoveries are leading to many new treatments for many conditions, mainly genetic conditions however. Nevertheless, I believe it is possible to use the newly available technology to treat viral and bacterial infections. In this essay, I will investigate to what extent can recent discoveries in the field of genetic and epigenetic editing impact the treatment of viral and bacterial infections.

Genetics vs Epigenetics

Genetics and Epigenetics are both very recently discovered fields and are still in the very early stages of development. With Genetics arguably starting with Gregor Mendel’s work with the common pea plant (Pisum sativum) and his discovery of the basic principles of inheritance in 1865. Despite such an early debut of the field, it advanced really slowly as better and better technology was needed to make new discoveries. On February 28 1953, James Watson and Francis Crick announced that they had discovered the structure of DNA (deoxyribonucleic acid) using X-ray crystallography (a technique in which the desired molecule is crystallised and placed in a synchrotron [machine which efficiently produces high intensity light {including X-rays}], once in the synchrotron the crystals are bombarded by high intensity X-rays from different angles. A metal film is placed behind the crystals to map the X-rays passing through the crystals. A model of the molecule can then be calculated using a complex computer program which takes into account both the X-rays which passed through the crystals and the X-rays which were reflected or absorbed). James Watson’s and Francis Crick’s discovery of the structure of DNA can also be argued to have been the beginning of genetics. There were also other very important discoveries which helped extend scientists’ understanding of the fundamentals of Genetics. Such as the discovery of the structure of the ribosome, reached using X-ray crystallography by 3 independent groups, led by Venkatraman Ramakrishnan, Ada Yonath and Thomas Steitz in the year 2000. Genetics has come a long way since and is still in its early stages of development.

Like Genetics, Epigenetics is also a very recently discovered field. However, Epigenetics is an even younger field that Genetics, with the term ‘Epigenetics’ being introduced in 1942 by embryologist Conrad Waddington who defined it as the ‘complex of developmental processes between the genotype and the phenotype’, and with studies labelled ‘Epigenetics only starting in the 21st century. There have been important discoveries in epigenetics, however none of them claim greater importance as the ‘fundamentals’ as Epigenetics has only recently begun being investigated by scientists. Some elements were also discovered a long time ago, however, science gained an understanding of their function only recently. For example, DNA methylation was discovered in 1948 by Rollin Hotchkiss. However, that was just the discovery of the modified cytosine itself, not its function nor what it was modified by and why. Epigenetics is, arguably, more complicated than Genetics, with many different molecules working together to create an environment, which makes it hard to label some as ‘more important’ or ‘fundamental’.

Genetics and Epigenetics are very similar, yet very different. They are intertwined, however both have different functions as concepts. Genetics is the study of genes and inheritance, whereas, Epigenetics is the study of gene expression and its consequences.

Genetics is the fundamental life science. It explores organisms’ genes and their change over time, inheritance and evolution. Genes are stretches of the medium an organism uses to store its genetic information (DNA or RNA), which each code for a specific protein. Both DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are made up of a sugar (ribose in RNA and deoxyribose in DNA) and phosphate backbone bound to nucleotide bases. There are 4 types of nucleotides (nucleotide bases) which are found in DNA molecules: Adenine (A), Thymine (T), Cytosine © and Guanine (G). In RNA, Thymine (T) is replaced by Uracil (U). Nucleotides can only bind to complementary nucleotides. Adenine binds to Thymine (in DNA) and Uracil (in RNA), forming 2 hydrogen bonds. While Cytosine binds to Guanine, forming 3 hydrogen bonds. Adenine and Guanine are larger molecules than Cytosine, Thymine and Uracil. Based on the size difference, nucleotides can be sorted into two groups. Adenine and Guanine are called purines, while Cytosine, Thymine and Uracil are called pyrimidines. The different combinations of nucleotide bases in an organism’s genes encode different proteins and form the genotype. Proteins are made up of amino acids. Each amino acid is encoded by a codon (a combination of 3 nucleotides). There are 20 amino acids and 64 different codons (4 { possible nucleotides} ^ 3 {nucleotides in a codon}). This leads to an abundance of codons. Hence, multiple codons can code for the same amino acid. There are also start and stop codons which signal the ribosome to start and stop translation when read, respectively. Exploring DNA and RNA, genetics focusses on the very fundamentals of life itself. Everything stems from genetics. The fundamental molecules which perform highly specialised tasks in organisms (proteins) are encoded by genetic material which is the main focus of genetics. Apart from genetic information, genetics also encompasses the transcription (from DNA to RNA) and translation (from RNA to protein) of genetic information. Many traits of the phenotype can be traced back to genetics such as eye colour, hair colour and much more complex things such as likelihood of becoming a nicotine addict. Many problems also stem from genetics. Phenotypical problems stemming from genetics are usually labelled ‘genetic disorders’. Genetic disorders can be less serious, such as colour blindness, or much, much more serious, such as sickle cell anaemia (for more details see chapter ‘Gene therapy’) or cystic fibrosis. However, some disorders labelled as ‘genetic disorders’ are actually not caused by genetics (specifically meaning the combination of nucleotides in one’s genes). Rather, the cause of the disorder can be epigenetic.

While genetics studies genetic information in the form of DNA and RNA, epigenetics studies how genetic information (mainly DNA) is modified. Epigenetic modifications to genetic information (mainly DNA) regulate gene expression. Gene expression is the name given to the phenomenon of transcription and translation a gene. Gene expression can have different levels and is usually used as a term signifying how much protein is produced from a given gene in a cell, tissue, organ, or organism. Epigenetics and genetics are intertwined as epigenetics is a concept stemming from genetics. Without genetic information, the epigenetic modification of genetic information to regulate gene expression would be impossible. Despite their similarities and intersections, they are two separate concepts and fields. There are many different types of epigenetic modifications. However, they can be sorted into two main categories: pre-transcriptional epigenetic modifications (chromatin modifications {chemical modifications to genetic information [mainly DNA] and the environment in close proximity of it}) and post-transcriptional epigenetic modification (RNA interference and degradation). Pre-transcriptional epigenetic modifications are mostly modifications to chromatin such as direct DNA modification or modification of histone protein octamers which DNA is bound to.

The main known type of direct DNA modification is DNA methylation. During the process of DNA methylation a DNA-methyltransferase enzyme catalyses the transfer of a methyl group to a cytosine nucleotide. This process usually occurs in chromatin regions known as CpG islands. In these regions, there are vast numbers of cytosine nucleotides directly followed by guanine nucleotides, hence the name C (cytosine) p (phosphate) G (guanine). A region is considered a CpG island if it is comprised of at least 200 base pairs, there is a CG percentage of greater than 50% and there is an observed-to-expected CpG ratio of greater than 60%. Cytosine nucleotides modified with a methyl group form a complex called 5-methylcytosine. The methyl marks on Cytosine nucleotides in the promoters of genes repress transcription of the gene and bump-down gene expression. This is because 5-methylcytosine inhibits the binding of transcription factors to DNA and recruits proteins involved in gene repression to further prevent transcription and translation of the gene. Unlike some other epigenetic modifications, DNA methylation is often found I region of the genome which are permanently silenced in the cell type. For example, a neuron doesn’t need to produce haemoglobin, hence the genes for haemoglobin need to be permanently silenced in the given cell type. DNA methylation is the main method for silencing genes in such scenarios as it a very strong repressive mark and is very hard to remove once added. However, if for some reason methylation needs to be removed from a certain nucleotide or region of DNA, its effects can be reversed by transferring a hydroxyl group to 5-methylcytosine. This reaction is also catalysed by a transferase enzyme. The reaction changes the structure of 5-methylcytosine in such a way which prevents DNA methyltransferase 1 (the main enzyme which maintains DNA methylation in humans) from binding to the DNA strand, hence DNA methylation cannot be maintained in the region and therefore the region can become epigenetically active.

Another pre-transcriptional epigenetic modification which influences gene expression is the modification of histone protein octamers which DNA is bound to. While DNA methylation is a strong repressive mark on gene expression, histone protein octamers can be modified to both silence and promote gene expression. Histone protein octamers are protein complexes with a core, consisting of 8 histone proteins, and 8 tails (1 coming off each histone protein). These tails interact with DNA and can be epigenetically modified to either promote or repress gene expression. There are 4 types of histone tails, they are as follows, H2A, H2B, H3, AND H4. Epigenetic modifications are mostly applied to only 2 types of tails — H3 and H4. There are two main types of epigenetic modifications which can be applied to histone octamer protein tails. The first is histone protein octamer tail acetylation, which is mostly associated with the promotion of gene expression. The second is histone protein octamer tail methylation, which is associated with both silencing and promotion of gene expression, depending on the precise position of the transferred group.

Histone (protein octamer tail) acetylation is a reaction where an acetyl group is transferred to an N-terminal histone (H3 or H4 not H2A or H2B) tail, the reaction is catalysed by a histone acetyltransferase enzyme. Histone acetylation is associated with promoting gene expression as it directly influences chromatin structure in the region. Histone protein octamers use their positively charged tails to bind to negatively charged DNA, maintaining chromatin integrity through strong electrostatic interaction. DNA bound to a histone octamer forms a structure called a nucleosome. When an H3 or H4 histone protein octamer tail is acetylated, its overall charge changes to neutral. This leads to severely weakened interactions with DNA. Due to weakened attraction between DNA and the n-terminal histone protein octamer tails, the DNA in that region unwinds to become less tightly packed and hence more available to transcription enzymes. This change in chromatin structure directly influences gene expression as more enzymes can get to the DNA in that region and transcribe it. Such regions of loosely-packaged chromatin are called euchromatin. Regions of euchromatin are usually transcriptionally active and have high gene expression.

Histone (protein octamer tail) methylation is a reaction where a methyl group is transferred to an N-terminal histone (H3 or H4 not H2A or H2B) tail, the reaction is catalysed by a histone methyltransferase enzyme. Unlike histone acetylation, histone methylation can be associated with both promoting and silencing gene expression, depending on the precise position of the transfered methyl group. There are two types of histone protein octamer tail methylation, one where the methyl group is transferred to an arginine residue on the histone protein octamer tail, the other where the methyl group is transferred to a lysine residue on the histone protein octamer tail. Both types of methylation predominantly occur on H3 and H4 histone tails. Arginine residue methylation is generally associated with enhanced gene expression, weakening the electrostatic interactions between the histone protein octamer tail and DNA and leading to the formation of euchromatin, and hence high levels of gene expression. Lysine residue methylation, however, can both enhance and repress gene expression, depending on the precise position of the transferred methyl group and the level of modification (mono-, di-, or tri-methylation of a single lysine residue). For example, dimethylation of lysine 9 on the H3 histone protein octamer tail (H3-K9), and trimethylation of lysine 27 on the H3 histone protein octamer tail (H3-K27) are generally associated with gene silencing and the formation of heterochromatin (tightly packed chromatin which makes DNA hard to access for transcription enzymes, reducing gene expression {opposite of euchromatin}). While, the methylation of lysine 4, lysine 36 , and lysine 79 on the H3 histone protein octamer tail (H3-K4, H3-K36, and H3-K79), is associated with promotion of gene expression and the formation of euchromatin. Unlike histone protein octamer tail acetylation, histone protein octamer tail methylation does not change the charge of the histone protein octamer tail. Rather, it serves as a mark for gene expression promoting or gene expression silencing associated enzymes which then alter chromatin structure in the region. Moreover, unlike histone protein octamer tail acetylation, histone protein octamer tail methylation is heritable (it is replicated during mitosis {cell division}), perhaps due to its less dynamic nature (histone protein octamer tail acetylation is edited very often), signalling a more mid-term role in gene expression patterns (DNA methylation being a long-term modification, and histone protein octamer tail acetylation being a short-term modification).

Histone protein octamer tail epigenetic modifications are much more complex than direct DNA modifications such as DNA methylation and can adjust gene expression more precisely. Nevertheless, there is another large category of epigenetic activity which can also potentially be very precise in regulating gene expression.

Post-transcriptional epigenetic activity, consists mainly of RNA and enzymes which regulate how much of the transcribed genetic material is translated. I shall focus on RNA which epigenetically regulates gene expression in (human) cells. There are two main categories which RNA molecules which regulate gene expression can be placed in. The first is long non-coding RNA molecules (lncRNA), the second is short RNA molecules. Both classes of RNA molecules which epigenetically regulate gene expression are non-coding RNA molecules. The term non-coding RNA means non-protein coding RNA, in other words, the RNA molecule can either not be translated at all, or the RNA molecule can be translated to form a completely dysfunctional protein.

Short RNA molecules are RNA molecules transcribed from regions of ‘junk DNA’ (DNA which doesn’t code for functional proteins) which are under 200 nucleotide bases in length. There are two main classes of naturally occurring short RNAs. Small interfering RNAs (siRNAs) and micro RNAs (miRNAs). Both types of RNA molecules regulate gene expression post transcriptionally by preventing translation of messenger RNA molecules. Small interfering RNAs interfere with translation of messenger RNA molecules by binding to a messenger RNA molecule with a complimentary nucleotide base sequence to that of the small interfering RNA, preventing the messenger RNA molecule from entering and being read by a ribosome (as the ribosome cannot read a double-stranded molecules {only single-stranded messenger RNA molecules}), preventing translation of the messenger RNA molecule. Micro RNAs are very similar in their ways of interfering with and preventing translation. Micro RNAs interfere with and disrupt translation of messenger RNA molecules by binding to a messenger RNA molecule with a complimentary nucleotide base sequence to that of the micro RNA, preventing the messenger RNA molecule from entering and being read by the ribosome and recruiting ribonucleases (RNA degrading enzymes) to break down the messenger RNA molecule into nucleotides. These mechanisms of epigenetic regulation of gene expression via short RNAs can be very precisely regulated, depending on the expression of the short RNAs themselves. If the ‘gene’ for the short RNA is a region of euchromatin with an abundancy of RNA polymerases and transcription factors, gene expression of the messenger RNA targeted by the short RNA is likely to be rather low. Whereas, if the ‘gene’ for the short RNA is in a region of heterochromatin with few transcription factors and RNA polymerases, gene expression of the messenger RNA targeted by the short RNA is likely to be rather high. The degree of expression of short RNAs can be very precise due to complex epigenetic histone modifications and chromatin structure in the region. This then makes for very precisely regulated gene expression of the target messenger RNA. Short RNAs can have quite a large impact on the expression of their target messenger RNA molecule, in a cell, potentially reducing the expression of the messenger RNA 100 fold, of what it would be if the messenger RNA were unaffected by the short RNAs.

Long non-coding RNA (lncRNA) molecules are (also) RNA molecules transcribed from regions of ‘junk DNA’ which exceed 200 nucleotide bases in length. Long non-coding RNAs can regulate gene expression both pre-transcriptionally, post-transcriptionally and inter-transcriptionally. Firstly, lncRNA can directly interact with DNA to alter chromatin structure in the region. This is one of the ways in which lncRNAs can epigenetically regulate gene expression and chromatin structure. lncRNAs can directly bind to DNA and alter its structure. This can lead to the formation of triple helixes (a shape very similar to a double helix but with an extra strand) or R-loops (a structure formed when and RNA molecule binds to one of the DNA strands and the other DNA strand is left in unwound form). By forming these structures, the lncRNAs can regulate the accessibility of the DNA to transcription enzymes such as RNA polymerases and other transcription factors. This can be used to both promote and silence gene expression in the region, depending on the structure formed and its accessibility to transcription molecular machinery. Secondly, lncRNAs can interact with various proteins. Many of these proteins can epigenetically regulate gene expression. For example, lncRNAs can recruit transcription machinery to promote the expression of a gene or region, or recruit DNA methyltransferases to silence the expression of a gene or region. Moreover, lncRNAs can interact with enzymes such as histone acetyltransferases and histone methyltransferases to create more complex gene expression patterns and chromatin structures. This can also be used to both promote and silence the expression of a gene or region. Both of these way of lncRNAs regulating gene expression are pre-transcriptional. lncRNAs can also influence gen expression patterns inter-transcriptionally. Due to their ability to interact with proteins, lncRNAs can interact with RNA polymerases and other transcription enzymes, apart from recruiting transcription enzymes to enhance gene expression, lncRNAs can also interfere with ongoing transcription by interacting with the associated enzymes to repress gene expression. Lastly, much like short RNAs, lncRNAs can epigenetically regulate gene expression post-transcriptionally. Due to their abilities to bind to RNA and interact with proteins, lncRNAs can interfere and prevent translation of messenger RNAs. lncRNAs can do so by binding to messenger RNAs with a complimentary nucleotide base sequence to that of the lncRNA, preventing the messenger from entering and being read by the ribosome and recruiting ribonucleases (RNA degrading enzymes) to break the messenger RNA molecule down into nucleotides. Much like short RNAs, lncRNAs can regulate gene expression very precisely, both promoting and repressing gene expression, according to the levels of expression of the lncRNAs themselves. However, unlike short RNAs, lncRNAs can be involved in long-term gene silencing. For example a lncRNA called Xist is essential for the inactivation of an X-chromosome in females (this is done to not overexpress certain genes as males only have 1 X-chromosome {with the male genotype being XY} while females have 2 X-chromosomes {with the female genotype being XX ). This silencing of an entire chromosome is seen in all female somatic cells (all cells other than germ cells) throughout the entire lifetime of the organism. This is a good example of the potential lncRNAs have for long-term gene silencing.

In conclusion, genetics and epigenetics are very different fields, which rely heavily on each other to function. Both of extreme importance to the phenotype and survival of an organism.

Viral infections

Despite common belief, viruses are not microorganisms. That is because viruses are not ‘living’. Viruses do not exist in the form of cells, but rather in the form of viral particles. Such particles consist of a capsid protein and the viral genetic information within the capsid. The capsid protein acts as a ‘shell’ for the viral RNA and replication enzymes within it. On the surface of the capsid, one may find antigen proteins, which allow the viral particle to bind to the receptor proteins on the surface of different cells. After the viral particle binds to a receptor protein on the surface of the cell, the viral RNA along with the RNA polymerases (replication enzymes {if present}) is transfered across the cell membrane into the cytoplasm. Once the viral RNA and RNA polymerases are in the cytoplasm the process of protein synthesis can take place. The RNA polymerases replicate the viral RNA and the virus uses the ribosomes of the host cell to translate its RNA. The synthesised viral proteins form a new viral particle. Once enough viral particles have accumulated in the host cell, the viral particles burst out in a process called lysis. Lysis kills the host cells and allows the replicated viral particles to infect other cells of the organism (if the organism is multicellular) or another organism (if the organism is unicellular or once a multicellular organism has died).

Some viruses such as HIV have a different method of reproduction. HIV is what is known as a retrovirus. Retroviruses are viruses which use reverse-transcriptase enzymes to reverse-transcribe their RNA into DNA. This mechanism allows the virus to change its genetic information to the same medium as the genetic material of the host cell. After the viral RNA is reverse-transcribed into single-stranded DNA, the virus uses DNA polymerases to synthesise a complementary DNA strand. Such sequences are then inserted into the host cell’s genome with the help of virus-encoded integrase enzymes. After the viral DNA is integrated into the host’s genome, the viral genes are transcribed alongside the genes of the host. The viral genes are translated into proteins, which then form new viral particles, however, retroviruses do not cause lysis and hence do not kill their host cell. Instead, the new viral particles pass through the cell membrane of the host cell and go on to further infect new cells. The integration of viral DNA into the host’s genome is extremely effective for retroviruses as the virus can continue to multiply until the host’s immune system detects abnormal activity from the infected cell and induces apoptosis (programmed cell death). This process of replication of viral particles can take place throughout the host’s entire lifetime if the condition is treated. This is because retroviruses are usually treated by immunosuppressant drugs to prevent the host’s immune system from destroying infected cells. The destruction of infected cells by the immune system is very dangerous and can be fatal for the host as if too many cells in a tissue are destroyed, it can cause decreased organ function and even organ failure.

As viral particles are not ‘living’ organisms, they cannot move around autonomously. Hence, they need a medium to travel through this medium is usually fluids produced by the host (for complex multicellular organisms). These include saliva, anal fluid, vaginal fluid, semen and blood. Hence the main ways of viral transmission are airborne and droplet transmission (saliva), transmission via sexual intercourse (anal fluid, vaginal fluid, semen), and transfusion transmission (blood). Viruses can also be transmitted by direct contact (touching a surface), however, this way of transmission is much easier to prevent as one can wash one’s hands and/or not touch mucus membranes after being in a public place, as that allows viral particles from the hands to enter the inside of the body and infect their target cell type. Airborne and droplet transmission is the most common way of viral transmission. Viruses such as influenza, SARS-cov2 and different types of rhinovirus (the common cold). Droplet viral transmission occurs when one’s saliva, contaminated with viral particles, exits one’s oral cavity, usually in the event of a sneeze or a cough and makes direct contact with a mucus membrane of another person. Airborne viral transmission is very similar as it is also reliant on an infected person’s saliva exiting their oral cavity, however, unlike in droplet transmission, the saliva droplets do not make direct contact with another person’s mucus membranes. Instead, the droplets rest on air or dust particles, using the air’s viscosity to maintain their level for a period of time (usually up to 3 hours), before descending to the ground due to their higher mass. During this period, the air or dust particles which the contaminated droplets rest on may be inhaled by a human. This leads to the viral particles, inside the droplets, entering the body and infecting their target cell type.

Viruses have an extremely high mutation rate. Approximately 1x10⁶ times higher than that of humans and 1x10³ times higher than that of bacteria. This is because viruses replicate much faster than humans and bacteria, which introduces many more random errors. Moreover, they lack a proof-reading mechanism for their genetic information after viral RNA replication. Because of this, it is very hard to make useful treatments for viruses as, often, by the time a new treatment is developed, the virus would have already mutated enough to render the treatment ineffective. Nevertheless, despite viral RNA constantly mutating, viruses have a part of the viral particle which stays constant in almost all cases — the capsid. This is because with a mutation in the antigens on the capsid, a virus would no longer be able to infect the same type of cells and hence many of its beneficial traits would, potentially, prove to be disadvantageous in a different type of host cell. Frequent changes in the capsid protein and the antigens on its surface would lead to lack of adaptation and specialisation among viruses and potentially to their extinction. Due to the capsid staying very similar between specimens of the same virus, it is possible to train the human immune system to recognise and destroy known viral particles. This is why vaccines are the most effective treatment for viruses. Once a virus has infected a host, it is hard to specifically treat the virus with conventional medicine, however vaccines are very effective at preventing infection and hence disease. Vaccines often introduce either a dead virus, attenuated virus or empty viral capsid (with no viral genetic information inside) into the body. This leads to a primary immune response where B-lymphocytes detect a pathogen in the bloodstream and produce antibodies to bind to the antigens on the surface of the capsid. The antibodies then, (by various ways) lead to the destruction of the viral particle. The genetic code for the antibodies is then stored by memory T-cells to be able to prevent infection by the same (this time harmful, unlike in the vaccine) virus, by inducing a vigorous secondary immune response if the virus is detected in the body.

Despite how effective vaccines are, there are cases when either an unvaccinated, or even (this happens very rarely) a vaccinated person is infected by a virus and is taken ill. In these situations, conventional medicine can, often, only provide supportive care (give the body required substances and maintain the best conditions for the person’s immune system to stop the virus form proliferating and killing cells). Unfortunately, at times this is not enough to keep the person in their best health. Nevertheless, I believe, recent advances in the field of genetic editing, can be used to create such therapies, which will counter viruses, even after the host organism has been infected.

Bacterial infections

Unlike viruses, bacteria are ‘living’, unicellular organisms and they can move autonomously. Moreover, unlike viruses most bacteria are not pathogenic. Nevertheless some bacteria are pathogenic and can cause much more serious symptoms in humans than viruses. Bacterial infections are transmitted in the same ways as viral infections. Nevertheless, bacteria are much more complicated in their mechanisms of entering different tissues and parasitizing cells. Because bacteria are ‘living’ microorganisms they need nutrients to survive. Pathogenic bacteria extract nutrients from their host, often causing weakness and less effective functioning of host cells. Pathogenic bacteria also negatively affect their host’s health by releasing toxins, such as botulinum and tetanus neurotoxins, which damage host cells and disrupt host cell function. The damaged host cells release chemicals such as histamine and bradykinin. These chemicals cause blood vessels to leak fluid into tissues, which causes swelling and inflammation. Bacterial infections can have much more serious consequences than viral infections, if left untreated. Nevertheless, most of the time bacterial infections are much easier to treat than viral infections.

Bacterial infections usually are harder and take longer for the human immune system to overcome, and hence cause a higher fever. Nevertheless, it is much easier to treat a human with a bacterial infection than a human with a viral infection (most of the time). Bacteria have a mutation rate approximately 1000 times lower than that of viruses. This makes developing drugs and treating people already infected by bacteria much easier than for people already infected by viruses. As most people know, bacterial infections are, generally, very efficiently treated with antibiotics. Antibiotics are substances which, selectively, either inhibit the proliferation or kill bacteria without damaging host cells. Antibiotics can be extracted from living organisms such as fungi or, artificially, chemically synthesised. There are two main types of antibiotics — bactericidal and bacteriostatic. Sometimes it is hard to classify an antibiotic into a distinct category. Nevertheless, the main distinctions are as follows. Bactericidal antibiotics kill bacteria, they may also inhibit the proliferation of the bacteria, however they must kill the bacterium. Bacteriostatic antibiotics inhibit the proliferation of the bacteria, they do not kill the bacteria. Many substances kill both host cells and bacteria, however, it is antibiotics that are used to treat bacterial infections. This is due to their ability to kill or inhibit the proliferation of bacteria, while leaving the host cells unharmed. This is possible due to sufficient distinctions between human cells and bacterial cells. For example, most bacteria have cell walls, while no animal cells do. This makes antibiotics such as the famous penicillin fatal to bacteria but harmless to human cells. Bacterial cell walls are partly composed of a macromolecule called peptidoglycan, while human cells do not synthesise or require peptidoglycan to function. Penicillin disrupts transpeptidation (the last cross-linking step in the assembly of a biological molecule) of peptidoglycan, leading to a very fragile, bacterial, cell wall, which then bursts killing the bacterial cell. However, the human host cells remain unaffected. Due to such distinctions between human and bacterial cells, antibiotics remain very effective against most bacteria. However, some bacteria have become less susceptible to antibiotics. This is slowly becoming a big problem for medicine and mankind.

Superbugs is the name given to species of pathogenic bacteria which have developed resistance to many types of antibiotics. Despite their much lower mutation rate compared to that of viruses, bacteria, like any organism bacteria also mutate. Some species of bacteria have mutated to develop a resistance against antibiotics. Using the previous example of penicillin, some bacteria have become resistant to penicillin. This has happened because of beneficial (for the bacteria) mutations in the genes which encode penicillin binding proteins. These proteins make up the active site of transpeptidase enzymes which catalyse transpeptidation (the reaction which penicillin disrupts). These mutations have deformed the penicillin binding proteins, making them no longer able to bind penicillin. By preventing the binding of penicillin to penicillin binding proteins, these mutations have given the transpeptidase enzymes the ability to bind substrates to their active site, and hence to catalyse the transpeptidation reaction once again. Beneficial mutations (for pathogenic bacteria) have affected many bacterial species and many of their genes, making them resistant to many different types of antibiotics. Such bacteria are becoming a big problem both for medicine and mankind in general. This is a big problem for medicine as some bacterial infections are becoming progressively harder to treat and some patients’ health can no longer be fully restored to its former state. As for mankind, as superbugs become more frequent and resistant to more types of antibiotics, we risk reaching a time, similar to how it was before antibiotics. A time when bacterial infections are extremely hard to treat and most bacterial infections are fatal. Nevertheless, I believe, recent advances in the field of genetic editing and virology, may solve the problem of superbugs and fully protect humanity from bacterial infections, once again.

CRISPR

CRISPR or Clustered Regularly Interspaced Palindromic Repeat is a palindromic, regularly interspaced (with other genes), repeating sequence of DNA, in the bacterial genome (different species of bacteria have different CRISPR sequences), which was first observed in the genome of E. coli in 1987. When these sequences were first observed, nobody even thought they were actually repeats of a gene, let alone what the potential of the protein they encoded was. Nobody payed much attention to CRISPR as it seemed like ‘junk DNA’ (the name given to DNA sequences not encoding a protein). Nevertheless, in 2012, teams led by Jennifer Doudna and Emmanuelle Charpentier, discovered the CRISPR cas-9 protein complex and its potential for genetic editing. CRISPR cas-9, originally, as other CRISPR proteins, evolved as a bacterial defence mechanism against viral infections. CRISPR cas-9 is often compared to ‘molecular scissors’, which is a simplified view of its function. CRISPR cas-9 uses a guide RNA molecule (bound to the REC1 domain), to bind to a specific section of DNA, cleave it, insert a DNA sequence and thereby (after DNA ligases catalyse the bonding between the nucleotides of the new DNA sequence and the nucleotides on the cut DNA ‘ends’), integrate it into the genome. CRISPR protects a bacteria from viruses by taking small fragments of viral DNA (either from a DNA virus or reverse-transcribed viral RNA) and inserting them into the bacterial genome after a virus has been eradicated from the bacterial cell. If the same (or similar) virus invades the bacterium again, the viral DNA fragments are transcribed, the resulting RNA molecules (both very small as transcribed from small DNA fragments) act as siRNAs (bind to an mRNA molecule and do not let it enter a ribosome for translation) or miRNAs (bind to mRNA molecules and make the mRNA a target for RNA degrading enzymes), thereby not letting the virus proliferate and slowly destroying the viral genetic information, conquering the viral infection. The same virus is also often memorised by other cells in the bacterial culture as bacteria can easily exchange genetic information through plasmids (independent rings of DNA, which can easily be transferred between cells). CRISPR does incredible things in bacteria and is therefore of great importance to bacterial cells. Nevertheless, CRISPR is gradually becoming of great importance to humans as it continues to do great things.

The CRISPR cas-9 protein complex, as previously mentioned, is now actively being used for genetic editing amongst different species and even different classes of life. CRISPR cas-9 is extremely useful for genetic editing due to its ability to, selectively, cleave DNA and then either ‘delete’ the cleaved fragment, replace it with a specific DNA fragment (artificially created in a lab), or insert a specific DNA fragment (artificially created in a lab) between the two cleaved DNA ends. Despite its high precision, CRISPR cas-9 is still not perfect. Science is not yet at a stage when one’s genome can be safely and reliably edited across multiple cell types and tissues. Firstly, CRISPR cas-9 is still not 100% accurate in its cleavage, deletion, insertion, or replacing of DNA. Secondly, it is very hard to ensure all cells in a tissue are edited, let alone an organ or the entire organism. In unicellular organisms such as bacteria, it is rather easy to perform genetic editing as there is only one cell, with one copy of the genome to edit. In very complex multicellular organisms such as humans, the number of cells to edit is incomparably larger. This leads to a higher probability of a mistake occurring (at least in one cell) and it makes it much harder to actually get the CRISPR cas-9 protein complex into cells. When working with a single cell, CRISPR cas-9 (along with the guide RNA) can be injected into the cell using a nanoneedle. Nevertheless, when working with multiple cells or a tissue, matters get much harder as immense amount of time is required to manually inject CRISPR cas-9 into the colossal number of cells in a tissue. Furthermore, if one is trying to edit all the cells in an internal tissue in a complex multicellular organism, one may no longer use a nanoneedle as it is near impossible to fit a needle through multiple layers of cells above the tissue and then inject CRISPR cas-9 into every cell. Lastly, if one is trying to genetically edit an entire organ or even an organism, things become even more complicated. When working with more than one cell, a vector is often used to deliver CRISPR cas-9 into the cell. Viral vectors are often very effective (the most common viral vector is an adeno associated virus), as viruses have evolved very reliable mechanisms to enter cells (because viral proliferation requires a host ribosome and hence viruses have to enter a host cell to reproduce {without such mechanisms viruses would cease to exist}). A viral vector is usually a viral particle with no genetic information (the viral genetic information is replace with the intended molecule {in this case CRISPR cas-9}). This allows the virus to deliver an intended molecule into the cell, without causing harm to the organism (due to no viral proliferation and hence no lysis occurring). Despite the efficiency of viral vectors, it gets progressively harder to reliably deliver intended molecules to cells, as the number of cells increases. Due to no viral proliferation, it is very hard to deliver enough viral particles (with intended molecules inside) to a tissue. Even if all the particles reach the intended tissue before getting detected and destroyed by the immune system, it is near impossible to precisely estimate the number of cells in the target tissue. This leads to unreliably (half) edited tissues. Such genetic editing attempts may not have the intended consequences and can sometimes end tragically for the organism. For example not uniformly edited tissues, with mistakes in the editing, can lead to apoptosis (programmed cell death) in the best case, and cancerous pathways being activated in cells throughout the tissue in the worst case. It has also been observed that CRISPR cas-9 can (in some cases) continue to make ‘collateral’ (unpredicted and unintended) edits to the genome, after it has edited the target DNA. Such ‘collateral’ edits can also lead to apoptosis or the activation of cancerous hallmarks in cells. This makes ‘collateral’ edits another major safety and reliability concern.

In addition to the problem of reliability, there is also the problem of ethics and that is a very big problem. Despite scientists’ great ambitions, genetic editing is limited by law. Most of the limitations of genetic editing are due to ethics as genetic editing is perceived by many as ‘playing God’. Many people are against genetically edited food, let alone genetically edited humans. Ethics is perhaps the biggest problem for genetic editing. The reliability problems of the genetic editing tools (such as CRISPR cas-9), will be solved as the field develops and experiments will be conducted to prove their reliability. Nevertheless, the problem of ethics will always be there (unless, perhaps, there is a crisis which threatens the existence of the human race and mass genetic editing is the only solution) as there are no fixed values for ethics and ethics are extremely subjective. In addition to the ethical question of whether one should be able to alter the very code which ‘made him’, there is also the safety and responsibility concerns. What if something goes wrong? Who is to be held responsible? Despite these ethical concerns, there already has been a case of genetic editing in humans. He Jiankui is a biophysics researcher from the People’s Republic of China who used CRISPR cas-9 to alter the genome of 2 human embryos. He used the genetic editing tool (the CRISPR cas-9 protein complex) with the intent of introducing a base pair deletion in the CCR5 receptor gene (CCR5 is a receptor on the surface of human white blood cells), to mimic the CCR5 delta 32 mutation which some people have naturally. This mutation introduces a premature stop codon into the gene and hence, once the mRNA is translated, the synthesised protein is faulty, resulting in a non-functioning receptor. This mutation is beneficial as CCr5 is the receptor which the Human Immunodeficiency Virus (HIV) uses to enter human white blood cells. The faulty version of the receptor synthesised from a delta 32 mutated CCR5 gene does not allow HIV to enter human white blood cells. Hence, the delta 32 mutation in the CCR5 receptor gene gives the carrier organism HIV resistance. He Jiankui attempted to grant the human embryos HIV resistance. Despite his well-intended genetic edits, he pleaded guilty and was sentenced to 3 years in prison on December the 30th 2020 (he, along with two collaborators had forged ethical review documents and mislead doctors into implanting human genetically edited embryos into two women). One of the women gave birth to twin girls in November 2018, the birth date of the other baby is unknown. I do not believe genetic editing should be performed in human embryos yet, however, I think it can be used in critical situations as a last resort. Such ‘critical situations’ include patients with late-stage genetic diseases which are nearly certain to be fatal without genetic editing intervention. I also hope that with the development of genetic editing technology, some restrictions will be lifted and scientists will have more freedom to edit the human genome, striving to eradicate genetic diseases (perhaps even some viral infections {like with the case of HIV}) and offer people a much better quality of life. In fact, genetic editing is already being used to treat some diseases in a developing branch of medicine called gene therapy.

Gene therapy

Gene therapy is a developing branch of medicine, still in its very early stages, where diseases are treated by either genetically or epigenetically editing cells. Both genetic editing and epigenetic editing are very newly discovered techniques and are still very much developing. However, genetic editing, for the time being, is much more reliable and safe than epigenetic editing. This is because epigenetics is an even more recently discovered branch of microbiology. There are still no ‘programmable’ enzymes which could reliably and safely edit the epigenome of a cell. There are, however, chemically synthesised drugs which have shown some results in epigenetic editing (changing gene expression) of cells. For now, genetic editing is more developed and hence has a higher potential to be used as a treatment for some diseases, especially genetic diseases. Unironically, genetic diseases, usually arise from mutations in the genome of an organism. Most mutations in one’s genome are harmless. This is because each protein coding gene, when transcribed into mRNA, has codons. Codons are blocks of 3 RNA bases (e.g. AUG), which code for amino acids. Because there are only 20 amino acids and 64 codons (4{possible bases}³{bases in a codon}), multiple codons code for the same amino acid. Due to this, if a mutation doesn’t change the amino acid which the codon codes for, the mutation is harmless as, after the mRNA is translated, the synthesised protein remains the same. For this reason, substitution mutations are often the most harmless as, one swapped base often leads to the codon coding for the same amino acid. Nevertheless, if the amino acid the codon codes for changes, the mRNA is often translated into a faulty (misfolded) protein. For this reason, deletion and insertion mutations can be harmful. This is because with an insertion or deletion mutation, the codons in a gene shift. With this shift, some codons change enough to code for different amino acids. Moreover, stop codons can be introduced prematurely, causing translation to stop earlier, synthesising a faulty, unfinished protein. A single faulty protein may not seem that bad. Nevertheless, mutations in the genome are there throughout one’s entire lifetime (unless corrected/changed by genetic editing). Moreover, some proteins are extremely important for organ and even organism function. For example, haemoglobin is vital for gas exchange, oxygen transport throughout the body, and respiration. Without haemoglobin, humans (and most complex multicellular organisms) cannot survive. For this reason, a substitution mutation in the haemoglobin-Beta gene on chromosome 11, causes a very serious disease called sickle cell anaemia. In this disease, haemoglobin is misfolded, which causes red blood cells to change shape from their natural biconcave shape to a sickle shape (hence the name sickle cell anaemia). This deformation of red blood cells is a very serious problem for the carrier of the mutation (with a homozygous recessive genotype) as, sickle-shaped red blood cells can often not fit into smaller blood vessels. Biconcave-shaped red blood cells can fold and hence can flow through even the smallest blood vessels. However, sickle-shaped red blood cells cannot fold and hence cannot fit through small blood vessels, not delivering enough oxygen to some tissues and often causing blood clots. Such blood clots can cause entire areas of the body to be starved of oxygen and can be fatal. Nevertheless, not all carriers of the mutation get symptoms of the disease. This is because sickle cell anaemia is what is called a recessive condition. In other words a homozygous recessive genotype (both the maternal copy of the haemoglobin-Beta allele and the paternal copy of the haemoglobin-Beta allele has to have the mutation) is required for symptoms to occur. If someone carries only one mutated allele, he is called a ‘carrier’ and doesn’t exhibit any symptoms. This is because in an organism with a heterozygous genotype for a recessive condition, the unaffected allele can compensate (be transcribed and translated much more to synthesise more unaffected proteins) for the mutated allele. There are also dominant disorders, in dominant disorders, such as Huntington disease, a heterozygous genotype (only one copy of the allele, a mutation in which causes the condition, has to carry a mutation) for the condition is enough for symptoms to show. In dominant conditions, the unaffected recessive allele cannot compensate for the mutated dominant allele as, the recessive allele is epigenetically shut down (gene expression {of the recessive allele} is stopped). Dominant conditions have no ‘carriers’ as the organism cannot have a mutated allele and stay unaffected.

Genetic disorders were always very hard to treat and have stayed that way. However, with recent advances in the field of genetic editing some experimental therapies have arisen, therapies which could stop genetic disorders from posing a fatal threat to an affected organism.

Gene therapy has already made its debut as a possible treatment. However, for now, most therapies are still in the stage of clinical trials. The main branches in which gene therapy (70 % of all ATMP studies) and other ATMP trials are ongoing [statistics from the UK, 2020] are oncology (35% of trials), ophthalmology (12% of trials), haematology (12% of trials), and metabolic (10% of trials). Despite these being the main areas with potential gene therapy treatments, there are many others such as cardiovascular (6% of trials), musculoskeletal (6% of trials), gastrointestinal (4% of trials), respiratory (2% of trials) neurological (2% of trials), and neuromuscular (2% of trials). [Catapult, n.d. The Cell and Gene Therapy Catapult UK clinical trials database. [Online] Available at: https://ct.catapult.org.uk/clinical-trials-database]

Some trials are attempting to use gene therapy to treat sickle cell anaemia and β-thalassaemia. Two main approaches have been taken on combating sickle cell anaemia and β-thalassaemia with gene therapy. However, both approaches manipulate the same gene, attempting to achieve the same result.

The first approach, taken by Vertex Pharmaceuticals (based in Boston) and CRISPR Therapeutics (based in Cambridge), uses CRISPR-cas9 to alter the BCL11A gene, in a way which renders the protein it encodes dysfunctional. The protein which BCL11A encodes, functions mainly as a transcriptional repressor and is essential for the epigenetic shutdown of the genes, encoding proteins required for the production of foetal haemoglobin. The protein encoded by BCL11A acts as an epigenetic ‘switch’, switching the production of foetal haemoglobin to the production of adult haemoglobin. Vertex Pharmaceuticals and CRISPR therapeutics are attempting to disable the protein encoded by BCL11A, thereby stopping the down regulation of the genes which encode the proteins required for the production of foetal haemoglobin. This flips the epigenetic ‘switch’ back to foetal haemoglobin, upregulating genes which encode proteins required for the production of foetal haemoglobin and down regulating the genes which encode proteins required for the production of adult haemoglobin. The main difference between foetal and adult haemoglobin is that foetal haemoglobin binds oxygen more strongly, this diminishes oxygen exchange in tissues, however, the difference is not critical. This ‘flipping of the epigenetic switch’ from adult haemoglobin back to foetal haemoglobin is not beneficial and perhaps even slightly disadvantageous for an unaffected organism. Nevertheless, for an organism with a genetic disorder such as those which cause sickle cell anaemia and β-thalassaemia, such a switch is extremely beneficial and potentially life-saving. This is because, in organisms affected by sickle cell anaemia or β-thalassaemia, there is a mutation in the haemoglobin beta gene (HBB), however, they do not have mutations in genes encoding proteins required for the production of foetal haemoglobin. Because of this, ‘flipping the epigenetic switch’ back to the production of foetal haemoglobin can be a very valid solution to their incredibly hard condition. The trials have shown that ‘so far, the participants with β-thalassaemia have not needed blood transfusions, and participants with sickle cell disease have not reported pain crises since the treatment’. This is incredible for the trials and hopefully could lead to a wider application of gene therapy in haematology and medicine in general.

The second approach, taken by researchers from Bluebird Bio (based in Cambridge) and led by haematologist David Williams at Boston Children’s Hospital, introduces a non-protein-coding gene into the genome of the affected organism’s blood stem cells, this is done using a lentiviral vector and most probably CRISPR-cas9 (not enough information was available on the study to state this for certain). This non-protein-coding gene codes for a short hairpin RNA, when transcribed. Short hairpin RNAs are artificially made RNAs, which can epigenetically regulate the expression of a target gene through RNA interference. RNA interference is a process when a short (< 200 nucleotides) non-protein coding RNA (such as a siRNA{small interfering RNA}, miRNA{micro RNA}, or shRNA{short hairpin RNA}) binds to a protein-coding mRNA molecule and ‘interferes’ with the translation of the mRNA by not letting it enter the ribosome (as there is now a second strand translation is impossible, the second strand acts as a ‘blocking’ strand) and, in some cases, attracting RNA -degrading enzymes such as ribonucleases, endonucleases, and exonucleases. When this non-protein coding gene is transcribed, it produces a short hairpin RNA which targets the same gene as the first trial (BCL11A), this short hairpin RNA downregulates the expression of BCL11A. The downregulation of BCL11A, once again, leads to the ‘flipping of the epigenetic switch’ back to foetal haemoglobin. The ‘flipping of the epigenetic switch’ upregulates the genes which code for proteins which are required for the production of foetal haemoglobin and downregulates the genes which code for the proteins which are required for the production of adult haemoglobin. After this switch, the organism produces a correctly folded (although different) form of haemoglobin, which hugely improves patients’ health and is potentially life-saving. This study has so far treated 9 participants with sickle cell anaemia.

The side effects from both of these studies consisting of infection and abdominal pain were temporary and linked to the treatments required to prepare the bone marrow for the procedure (not the actual gene therapy). This inspires much hope, as with improvements of the method of delivery of the biological molecules to the target cells, the side effects may become less and less serious, potentially even fully disappearing. For now, such trials are limited to people with very severe disease as the procedures are hard to perform and have not yet reached maximum safety. However, I believe and hope gene therapy will improve and become more widely available as it could truly work wonders for modern medicine, this is especially true, now, after we have gotten a glimpse of gene therapy’s potential in clinical studies.

Despite the many positives of gene therapy, there are, for now, some rare, however, very alarming negative effects.

Recently, Bluebird Bio had to halt another gene therapy trial for sickle cell disease as two of the participants developed ‘leukaemia-like’ cancer. This trial used an adeno associated virus (a virus related to HIV) to insert a non-mutated copy of the HBB gene into the genome of the participant organisms. This unmutated copy of the gene is meant to compensate for the organism’s two mutated copies, producing correctly folded haemoglobin after translation and therefore allowing the organism to decrease symptoms and become healthier. Nevertheless, despite the good intentions two of the participants developed ‘leukaemia-like’ cancer after the treatment. This could have happened for multiple reasons. Firstly, there could be a problem with the gene itself or its insertion {perhaps the region of the genome where it was inserted} (genetic therapy part of the treatment), there could be a problem with a adeno associated viral vector {perhaps the virus inserting its genetic information into the participants’ genome} (the delivery of the biological molecules to the cells), or there could be a problem with the chemotherapy given to the patients to kill the remaining bone marrow cells before the treated bone marrow cells are given back to the participant (the treatment is performed ex-vivo). The viral vector had indeed inserted its genetic information into the participants’ genome, however, its exact location in the genome is still unknown. Despite this information, the researchers suggested it was most likely the chemotherapy given to the patients, which had caused this leukaemia-like cancer by damaging the participants’ DNA and triggering cancerous pathways. This is potentially quite alarming and could be devastating for the field of genetic editing. On the day Bluebird Bio halted the sales of its already approved treatment in Europe, the company’s stock plunged by an astonishing 38%. I truly hope this will not have a major effect on the field of genetic and epigenetic therapy. Such a development is devastating both for the patients and for the field, because of common opinion towards genetic and epigenetic therapy. It really brings out people’s old fears and could lead to a tumbling reputation for the field. People are often blinded by fear, and it is then, that they fail to see true potential and make the worst decisions. I hope, however, scientists actively working in the field will prove and improve the safety of gene therapy and avoid any new governmental restrictions imposed on genetic and epigenetic therapies. Despite these terrible developments in this study, 14 people treated with this method by Bluebird Bio are now ‘virtually free of the pain crises their sickled red blood cells once caused’.

Gene therapy is still in its very early stages and there is much room for improvement, however, it can be the solution for many problems and a cure for many diseases, as it has already shown. For improvements to be made and for the field to advance, people need to believe in its potential and sometimes, perhaps even, overlook extremely unfortunate setbacks such as the one mentioned earlier. Hopefully they will be very rare, however, every technology has setbacks during development. At times it is hard to go on after such events, however, one must remember the good intentions of the research and treatment of the patients and empathise with both the scientists and the patients, as after such events, both groups have to experience extreme hardships.

As gene therapy will continue to advance and progress, some diseases may even be eradicated. I hope someday we will be at a stage when nearly any disease could/would be treated with gene therapy. I am not only talking about genetic disorders, but also viral and microbial infections.

Gene therapy to combat viral and bacterial infections

Viral infections can be negligible to a human organism (such as the rhinoviruses which cause the common cold) or can be very serious (such as HIV or Ebola). At the moment, viral infections are hard to treat when the host human organism has already been infected. It is easier to develop a vaccine to prevent the infection, rather than a post-infection treatment . This is due to viruses’ extremely high mutation rate, likely rendering a treatment useless soon after development (for a more detailed explanation see chapter ‘Viral Infections’). However, I believe gene therapy can be used to treat viral infections post-infection and pre-infection.

Once a virus has invaded a host cell it uses the host’s ribosomes to translate the viral genetic information (usually RNA). The proteins produced after translation then go on to form new viral particles and invade new cells (for more details see chapter ‘Viral Infections’), often killing the host cell in the process. Viral replication can be stopped by gene therapy. There are two main ways (both gene therapy) to stop viral replication and proliferation post-infection. One way is to use short RNA and RNA interference to hinder or fully stop the translation of viral RNA. The second way is to use CRISPR-cas13 to cleave viral RNA in the host cell before it is translated, the cleavage would lead to faulty viral proteins being produced and hence would hinder or fully stop viral replication and proliferation post-infection.

Using short RNAs to prevent viral replication and proliferation is a multis-step process which requires a vector, a short RNA which targets a certain viral gene and a subsequently derived gene for the short RNA. The short RNA can be delivered to the cells of the subject organism using a viral vector. The viral vector should deliver a CRISPR-cas9 protein complex with some target DNA (which when transcribed produces the short RNA) and perhaps some short RNAs targeting the virus (especially if the treatment is delivered post-infection). Once inside the cell, the CRISPR-cas9 protein complex should insert the target DNA into a target region. The short RNAs delivered by the vector would be effective in post-infection treatment as they would make for an immediate response to the viral RNA already in the cytoplasm. The target DNA inserted into the organism’s genome by the CRISPR-cas9 protein complex would serve as a more long-term solution to the virus. When the target DNA sequence is expressed, the short RNAs are produced. The short RNAs then go on to bind to the viral RNA, preventing it from entering the organism’s ribosomes and recruiting enzymes to degrade the viral RNA. This hinders or potentially even completely stops the process of production of a target viral protein, hindering or completely stopping viral replication and proliferation. The antigen proteins on the capsid would be a good choice for a target protein as they do not change (due to mutations) nearly as often as other viral proteins (for more details see chapter ‘Viral infections’). This method of gene therapy to counter viral infections could both prevent infection and counter the viral pathogen post-infection.

CRISPR-cas13 is very similar to CRISPR-cas9. The main difference between the two is the nucleic acids which they are able to cleave. CRISPR-cas9 is able to cleave DNA, whereas CRISPR-cas13 is able to cleave RNA. This (potentially) makes the CRISPR-cas13 protein complex extremely useful in combating viral replication and proliferation. CRISPR-cas13 also uses a guide RNA sequence of around 30 nucleotides (just like CRISPR-cas9), to bind to an RNA molecule {usually a messenger RNA} (unlike CRISPR-cas9 which binds to DNA), after which the protein complex cleaves the sequence complimentary to that of the guide RNA from the other RNA molecule. This allows CRISPR-cas13 to bind to and cleave specifically targeted viral RNA molecules, hindering or completely shutting down translation of the viral protein the target viral RNA molecule codes for, hindering or completely disrupting the process of viral replication and proliferation. CRISPR-cas13 has been used experimentally in labs, however, it has not been used in vivo due to an alarming trait. CRISPR-cas13 has been found to be prone to ‘collateral cleavage’. ‘Collateral cleavage’ is the name given to a phenomenon in which CRISPR-cas13 (after it has cleaved the target RNA {according with the guide RNA}) cleaves off-target, non-marked RNA molecules. This can have a wide range of effects, ranging from negligible under expression of a gene to potentially activating cancerous pathways. ‘Collateral cleavage’ is a very unpredictable phenomenon. If the ‘collaterally cleaved’ RNA is a small interfering or micro RNA in the cytoplasm which hasn’t bound to an mRNA, the consequences can be negligible, such as, an extremely slight over expression of a gene. However, if the ‘collaterally cleaved’ RNA is a messenger RNA coding for a vital enzyme or hormone, the consequences can be more severe, such as, it might (even if only slightly) affect the function of a tissue. In the worst possible scenario, the ‘collateral cleavage’ affects messenger RNA which codes for vital proteins and it is cleaved consistently. If a certain messenger RNA molecule is consistently cleaved, the expression of the gene may be impacted significantly, which can be harmful to the organism, especially if the messenger RNA cleaved codes for a vital protein. Despite this phenomenon of CRISPR-cas13, I believe it can still be very useful for post-infection treatment of viral infections. With further advances in the field, the protein complex will likely be edited and perfected to be made more safe and reliable. However, even after these changes are made, CRISPR-cas13 will only be useful for post-infection treatment of viral infections as the protein complex will eventually be degraded by the cell after a certain time period.

Another major drawback for gene therapy in general is the delivery of the biomolecular machinery to the target cells. However, like many aspects of gene therapy, the technology is still in its very early stages and improvements will be made as the field progresses.

It is harder to prevent bacterial infections, however, it is easier to treat them post-infection. The main way for treating bacterial infections post-infection today is through the use of antibiotics. However, superbugs (bacteria resistant to the effects of antibiotics) are becoming increasingly problematic (for more details see chapter ‘Bacterial infections’). Gene therap0y has the potential to be the solution to this problem. There are two main ways in which gene therapy can be used to counter bacterial infections post-infection. One way is to use bacteriophages to invade and kill the host bacterium. The other way is to deliver genetic information coding either for harmful proteins or for translation silencing RNAs into the target bacterium, killing the target bacterium either through intoxication or inability to produce vital proteins.

Bacteriophages are a major class of virus which infect bacteria. Bacteriophages can be very effective in treating bacterial infections as they have evolved to invade, multiply in and kill bacteria. Similar to viruses which infect humans, lytic phage for example, disrupt the metabolism of their host bacterium and cause it to lyse. Bacteriophages were tested in humans infected by bacteria as far back as 1919. However, bacteriophages never became a widespread therapeutic method as antibiotics were discovered, which were easier to produce, store and use. Antibiotics were extremely effective in countering bacterial infections and became the worldwide solution to bacterial infections. Nevertheless, with the extensive use and overuse of antibiotics, many strains of bacteria started becoming less and less sensitive to antibiotics, developing resistance and earning themselves the name ‘superbugs’ (for more details see chapter ‘Bacterial infections’). Superbugs are a problem now more than ever. Bacteriophages may be the potential solution. Due to their high effectiveness in infecting and killing the host bacterium, bacteriophages may become the ‘new antibiotics’, being used worldwide to treat bacterial infections. However, there is concerning property of bacteriophages which could potentially make them harmful to human cells as well. Transduction is the process of bacteriophages integrating part of the host bacterium’s genome into their own and passing it on to the next host bacterium. This can occur both by fragments of DNA or plasmids entering a newly created bacteriophage particle by chance, or through the bacteriophage copying some bacterial genes (close to the point of insertion of the bacteriophage genome in the bacterium’s genome) along with its own genetic information, before packaging it into a new bacteriophage particle. After lysis (usually), when the new bacteriophage particles go on to infect new bacteria, the transduced bacterial genes are inserted into the new host bacterium’s genome. This may have negative consequences for human cells as the transduced genes may be genes which promote further antibiotic resistance or a gene for another toxin, strengthening the bacteria. Nevertheless, with advances in the field, improvements to the technology will be made, the treatments will be refined and made more safe and reliable, with bacteriophages potentially becoming the new treatment for bacterial infections to work alongside or fully replace antibiotics.

Delivering CRISPR-cas9 with harmful genetic information to insert into the bacterial genome may also be a viable option. One would need a (viral) vector, potentially using a recombinant bacteriophage to deliver the CRISPR-cas9 protein complex along with the genetic information to the target bacteria. After which, the CRISPR-cas9 protein complex would insert the genetic information into the bacteria’s genome. The genes delivered should code for RNA digesting enzymes and short RNAs and potentially genes for proteins such as galectin-8 (encoded by the LGALS8 gene) which kills intracellular bacteria. With the expression of the target genes alongside the bacterial genes the bacteria would be ‘digging their own grave’, as with the expression of the target genes, bacterial transcription and translation would be heavily disrupted hindering or potentially completely shutting down bacterial replication and proliferation, killing the bacteria through lack of vital proteins. Moreover, the genes for harmful proteins such as galectin-8 would recruit autophagy adaptors, leading to the formation of an autophagosome and the subsequent destruction of the intracellular bacteria. The main dangers and drawbacks of this approach would be the delivery of the material to the host bacteria as a lot of material is required for the therapy, the extremely small risk of ‘collateral’ CRISPR-cas9 cleavage, and perhaps the most concerning one, is the possible heavy disruption of transcription and translation in human cells as well as bacteria leading to apoptosis and potential tissue damage. However, with the development of the field, the therapy will be edited and refined to become more safe and reliable. This could potentially become another valid way of treating bacterial infections (especially severe bacterial infections).

In conclusion, gene therapy is still in its very early stages and still has many risks and unreliabilities. However, with advances in the field, the methods will be edited and refined to make for a more reliable and safe way of treating many diseases. Genes code for the fundamental molecules required for life and the creation of cells and organisms, a change in those fundament molecules can be both extremely beneficial and devastating for the organism. Humanity now has the tools to change these fundamental processes and molecules and use them to their advantage. I believe gene therapy has the potential to treat nearly all diseases and do so with colossal superiority to other treatments humanity has to this day.

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