MAYA ACHUTHAN
Age 15 | Victoria, BC
2017, 2018, 2021 Genome Alberta Award; 2018, 2021 ERSF Silver Medalist | 2021 Edmonton Society of Gastroenterology Human Biology Award | 2020 YSC Online Fair Regional Award | 2021 CWSF Bronze Excellency Medal
Edited by Nico Werschler
INTRODUCTION
Cystic fibrosis (CF) is a rare, chronic, progressive, and frequently fatal genetic disease of the body’s mucus glands. Although CF is a multi-organ and multi-system disease, it primarily affects the respiratory and digestive systems in children and young adults. According to the data collected by the Cystic Fibrosis Foundation, there are about 30,000 Americans, 3,000 Canadians, and 20,000 Europeans with CF (Cystic Fibrosis Foundation, n.d.). Approximately 1 in 2,500 babies are born with CF each year in the United States. And about 1 in every 20 North Americans is an unaffected carrier of an abnormal “CF gene.” There are around 12 million people who are usually unaware that they are carriers (Centers for Disease Control and Prevention, 1995).
Cystic Fibrosis (CF) is a disorder that is produced by mutations within the Cystic Fibrosis Transmembrane Regulator (CFTR) gene (Vaidyanathan et al., 2020). The complexity of CF is that the pathological patterns vary between patients and in varying intensity. However, the basic problem is the same - an abnormality in the glands, which produce or secrete sweat and mucus. In healthy patients, the CFTR protein (which is an ion channel, moves electrically charged atoms/molecules back and forth through the cell) moves chloride ions at the regular pace, from the inside to outside of the cell, allowing cilia movement (Cystic Fibrosis Foundation, n.d.). Due to the mutations, chloride ions are trapped inside cells, preventing water from flowing through the cell. Due to water insufficiencies, regular mucosal levels rise and become more adhesive. Thicker mucus decelerates the speed of cilia movement. If the cilia no longer move at regular speeds, mucus begins to fill up airways and bacteria reproduce (Cystic Fibrosis Foundation, 2019). Mucus in CF patients accumulates in the intestines, pancreas and lungs resulting in malnutrition, poor growth, frequent respiratory infections, breathing difficulties, and eventually permanent lung damage. CF disease progression is cyclic: lung infections, inflammation/pulmonary exacerbations, and added mucosal buildup (CF Source, n.d.). Improved medical technology has allowed for extremely advanced infantile screening technology; recent studies from 2019 predict that infants testing positive for Cystic Fibrosis have a lifetime expectancy of at least 48 years. Bronchiectasis (lung failure) is the usual cause of death in most patients (Cystic Fibrosis Foundation, 2019).
Since airway inflammation is generally very high in CF patients, certain measures should be taken before actually administering the following therapy. Drugs and technology should be utilized to create the optimal environment for cellular and genetic editing.
Figure 1: Most common CF causing CFTR sequence, Delta F508, versus healthy CFTR sequence.
Though there are many treatments for suppression of CF symptoms and disease progression, it is speculated that gene therapy may be the only cure. Given the disorder is monogenic (controlled by a single gene), this research explores the options, applications, and delivery of CF lung treatment via a CRISPR-Cas9 procedure.
BACKGROUND
CRISPR is an acronym for Clusters of Regularly Interspaced Short Palindromic Repeats. It is a specialized section of DNA with two distinctive characteristics: a repeated arrangement of nucleotides and spacers. Nucleotides are known as the basic building block of polymers, such as DNA or RNA. A single nucleotide consists of two main parts: (1) a sugar molecule and phosphate group (makes up the sugar phosphate backbone) and (2) a nitrogen base. DNA is made of bases; adenine, guanine, cytosine, and thymine, while RNA is made of bases; adenine, guanine, cytosine, and uracil. Spacers are usually found on chromosomes and serve the purpose of separating active sections of DNA and do not have any coding or transcription properties. Cas9 is a type of enzyme that can separate sections of DNA. It is this enzyme that separates the genetic material and allows for geneticists to biologically remove the mutation.
When a virus (or disease) tries to invade the body, sections of mutational DNA are transcribed into the spacer. This combination of spacer and DNA generates a type of RNA specific to CRISPR: crRNA. Transcription is the process in which genetic information is copied into a molecule of RNA, usually messenger RNA (mRNA). Then, the crRNAs are used as template to guide the Cas9 enzyme specifically to the mutated section of the DNA. The sgRNA molecule is a product of the crRNA and tracrRNA (which effectively activates it).
Figure 2: The natural CRISPR-based process within bacteria
This gene-editing procedure is derived from a naturally occurring phenomenon in various bacterial species (for example, E. Coli) against phage attacks. Pieces of DNA from the invading viruses are collected and saved by the bacterial species. The fragments of DNA are then used to ‘disable’ the virus as it invades the host. The ability for geneticists to mimic the bacteria’s unique behaviour in laboratories places precise gene-editing via CRISPR-CAS9 on a higher pedestal than ever before.
Although CRISPR-Cas9 applications present as promising solutions to treat and cure genetic disorders, major setbacks must be considered. Arguably, delivery of the CRISPR-CAS9 molecule targeting specific affected organs itself is the biggest challenge scientists are facing (Behr, Zhou, Xu & Zhang, 2021). Currently, the use of adeno-associated viruses (AAVs) to transport CRISPR are heavily relied on, in vitro (outside the body; in a laboratory). This, although heavily aids the race to find a CRISPR-based solution for various diseases, does not provide a feasible solution for in-vivo procedures. In other words, how we can deliver the CRISPR molecule within the body, without the need to transplant cells from and to the patient.
There are six main CFTR mutation types in patients that suffer from CF. The F508del mutation is the most prevalent among cystic fibrosis patients. Due to this mutation, a single amino acid is removed from the CFTR gene, and that causes the CFTR protein to be synthesized incorrectly and cannot function properly. The CFTR protein oversees the maintenance of chloride (salt)/water balances in the body. When this protein is not synthesized properly, chloride becomes trapped in cells. Without movement of chloride, the body does not stay hydrated, and as a product the naturally occurring mucus becomes thicker and stickier. The body recognizes this mistake and does not produce the protein. CRISPR-Cas9-based gene-editing methods are being tested among scientists to fix this mutation.
Now, applying this technique to the affected lungs in CF patients, specifically to the CFTR affected epithelial cells of the bronchial region requires to factor the following challenges:
Editing technology degradation (Chow et al., 2020)
Rapid renal clearance (Chow et al., 2020)
Efficient movement and deposition to the affected organs (Chow et al., 2020)
Movement and deposition of gene-editing complexes (CRISPR) to the alveolar region of the lungs instead of the bronchial region (Chow et al., 2020)
Gene transfer into the lung is difficult due to extracellular barriers (mucus, mucociliary clearance, immune responses, airway inflammation) and intracellular barriers (nuclear membrane) (Chow et al., 2020). The greatest obstacle lay in the deliverance of effective gene therapy solutions. Currently, research proposes the transportation of the CRISPR-Cas9 molecule to the epithelial cells of the bronchial region in the form of an inhalable aerosol (Chow, Chang & Chan, 2021).
MATERIALS & METHODS
Orally inhalable aerosols would bypass a few of the current problems surrounding traditional CRISPR delivery methods. Improved genes are sent to target cells quickly and efficiently, at high concentrations and with the least amount of systemic exposure possible (Chow et al., 2020). This moderately ‘local’ delivery of the CRISPR molecule allows for the non-intrusive access to the respiratory system, more specifically, the lower respiratory system.
For any drug to be distributed through inhalation, it needs to be in either solid or liquid aerosol form. An aerosol is a liquid or solid mixture suspended in air, consisting of either small or large droplets (Morgenstern, 2020). An aerodynamic diameter is the spherical diameter of a single droplet within an aerosol with a particle density of 1g cm−3. It is the general idea that aerosol droplets/particles with an aerodynamic diameter of 1 to 5 μm are technically acceptable for use in pulmonary aerosol delivery, but it was later discovered that aerodynamic diameter of 2 to 3 μm provides the most substantial therapeutic benefit to patients (Edwards et al., 1998). This is because, especially in minimized airways, smaller particles generally have a lower deposition rate. When transporting gene editing enzymes, however, larger size is needed to contain the enzyme/genetic information. 2 to 3 μm fits both criteria.
RESEARCH
The research has identified the following three main ‘hurdles’, or biological barriers, that the inhalable aerosol should address to effectively enter the nucleus of the epithelial cell in the bronchi:
Constricted Airways
Airway Surface Liquid
Distribution to Bronchi Epithelium
The sections below will explain the above challenges and theoretical design elements of the aerosol to bypass and address these physiological as well as anatomical blockades.
Figure 3: Cross section of a CF bronchial epithelium highlighting the various blockages involved.
RESULTS
1.0 Constricted Airways
The lung is biramous; it diverges into two sections. Cystic fibrosis causes mucus-constricted airways, leading to an extremely infectious and inflamed environment within the lung (Chow et al., 2020). Due to this, the movement of the aerosol to the lower respiratory tract is blocked.
The two key characteristics of aerosol movement are the physical attributes of the particle and the basic gas-flow dynamics within the lung.
The ability to transport a given particle relies on its size. To calculate the movement of the particle, the particle diameter and mean free path of the air with the lung need to be considered. Mean free path is the average distance between collisions that a gas molecule travels (The mean free path of air at room temperature (atmospheric pressure: 1013 hPa) is 68nm, or 0.068 μm.
Remembering that the diameter of the particle providing the most therapeutic benefit is 2-3μm (Edwards et al., 1998) (average: 2.5μm), we find the ratio of diameter to mean free path to be 2.5/0.068. The quotient of this ratio is equivalent to a term called the Knudsen number (Kn).
The Knudsen number generated is 36.8 (36.7647058824). Since the Knudsen number is higher than one, the particle's hydrodynamic flow follows the Knudsen regime. Further, any particle that oversteps a measurement of Kn ≃ 7 follows free molecular regime. This basically means that the presence of the particle itself does not affect the surrounding gases in a given container, which is the lung in this case.
It is understood that monodisperse aerosols (aerosols with equal sized particles) have lesser deposition rates than using polydisperse aerosols (particles vary in size).
The deposition of a particle can be based on two defined parameters, emitted dose (ED) and fine particle fraction (FPF). Emitted dose is the total amount of particles that initially leave the inhaler, and fine particle fraction accounts for any particle that has an aerodynamic diameter of less than 5.8μm. FPF shares an inverse correlation with deposition rate (i.e., higher FPF = lower deposition rate, and vice versa).
A study was done to track which particle shape had a higher FPF percentage. The actual shape of the particle directly impacts its consequent aerodynamic motions. The types of particles studied were:
Pollen-shaped hydroxyapatite particles
Spherical hydroxyapatite particles
Spherical calcium carbonate particles
Plate-shaped calcium oxalate particles
Cube-shaped CaCO3 particles
Needle-shaped CaCO3 particles
Pollen-shaped had the highest FPF percentage. However, the particles had an aerodynamic diameter higher than 2-3μm. If particles with a smaller aerodynamic diameter (fitting into the therapeutically beneficial parameters) could be made into a pollen-like shape, we could reach better efficiency concerning premature respiratory deposition.
In terms of particle density, it was discovered that particles with a smaller diameter and greater density had higher deposition rates than slightly larger particles with lower densities (Toy et al., 2011). This is because smaller particles generally deposit within lung cavities by a process called Brownian Diffusion; when particles deposit due to their collisions with existing gas particles, making their movement irregular, increasing general diffusion rates. Brownian Diffusion is influenced by particle size, but conveniently not density: the smaller the particle, the higher the diffusion rates. For many therapies, early deposition is beneficial, but for bronchial epithelial cell editing, lower early deposition rates should be gained.
1.1 Outcome: Constricted Airways
Based on the above assessment, to address the constricted airway of the lungs, aerosol particles should be designed as follows:
The particles should have a diameter between 2 and 3μm.
The particles should be monodispersed and pollen shaped.
The particles should be manufactured with the lowest amount of particle density possible.
2.0 Airway Surface Liquid
The abnormal CF mucus is a by-product of mutations within the CFTR gene. It consists of a gel-type mucin fiber that is made up of many negatively charged macromolecules and is a barrier between the aerosol and the cells (Chow et al., 2020).
Scientists have overcome the mucus barrier through the engineering of polymeric gene carriers that are tiny enough to pass through the framework of the mucus (Suk et al., 2013).
Cationic polymers (a type of nano- or micro- particle) possess a high density of protonable amines, which are neutralized ammonia-based organic compounds, that facilitate:
Effectively condensation of DNA (Sun & Zhang, 2010; Dunlap et al. 1997)
Protection of cargo DNA from enzymatic degradation from mucus (Kukowska-Latallo et al., 2004; Ferrari, 1999)
Endosomal escape of cargo DNA (way of transport into cell) is possible through cationic polymers (Akinc et al., 2004)
Between the two cationic polymers tested for ability to penetrate the mucus layer in bronchial airways, polyethylenimine (PEI) and poly-l-lysine (PLL), polyethylenimine has the higher transfection rate*. The transfection rate for PEI is 45.5% transfection efficiency versus 36.8% transfection efficiency for PLL (Wang, 2019).
*Transfection efficiency is a measure of how easily the particle can transfer the genetic information (CRISPR-Cas9) to the affected cells.
Previous studies have shown that coating of a special type of compound polyether, called Polyethylene glycol (PEG), allowed for cervicovaginal mucus penetration.
A compound polyether is a type of substance created by polymerizing simple compounds, also known as monomers (Britannica, 1998). This polyether is frequently used in biomedical therapies because of its exceptional biocompatibility, bioinert stability, resistance to protein absorption, and non-immunogenicity (Zhu, 2010). PEG is created via the polymerization of ethylene oxide through a very specific technique called ring-opening. This technique allows the synthetization of PEGs of varied weights and weight distributions (Moore, 2020). PEGs are frequently used as a particle coating for drug delivery vectors for three reasons:
PEGs prevent opsonization. Opsonization is the process by which opsonin proteins cohere to microparticles, allowing the body’s natural defense system (immune system responses and macrophages) to effectively remove the particle.
PEGs prevent phagocytosis. Phagocytosis is the process where cells called phagocytes take in or ‘ingest’ microparticles or invading cells as a defence reaction.
PEGs prevent aggregation. microparticle aggregation is the tendency of microparticles, in some circumstances, to aggregate or bunch together to form clusters. PEG-coated microparticles change to negatively charged and acidic particles.
Uncoated PEI polymers are called PEI-UCP. PEG-coated PEI polymers are called PEI-CCP. Researchers found that a cationic polymer created with 25% PEI and 75%
PEG5k –PEI (called mucus-penetrating-polymers, or PEI-MPP from now onwards) were:
Extremely compacted while maintaining small hydrodynamic diameters: ~50 nm (equal to 0.05μm)
Near neutral in surface charge comparable to PEI-CCP, due to efficient PEG coating
PEI-MPP displayed an improved stability compared to PEI-CCP (Suk et al., 2013)
PEI-CCP exhibited lower DNA compaction stability in presence of anionic heparin than PEI-UCP (as expected due to the lowered number of amines and steric hindrance by PEG chains)
Both PEI-CCP and PEI-MPP protected cargo DNA as efficiently as the uncoated PEIs throughout mucus penetration.
When tested through mouse airways, the PEI-CCP had aggregated and formed clusters, effectively depositing in the airways (Suk et al., 2013). After a period of six hours after drug administration, the PEI-CCP had dropped to around 30% of the original drug given (due to mucosal clearance), while PEI-MPP had retained around 70% deposition in the lungs.
The use of PEI-MPPs to transport genetic material past the mucosal layer did not trigger immune reactions from the body. This means that aside from the natural inflammation that is a product of bacteria found in mucus in CF patients, no extra inflammation occurred as a product of the cationic polymer used.
A polyplex is the combination of a cationic polymer (PEI) and a DNA plasmid (material that holds the CRISPR-Cas9 information).
Mucus is negatively charged. Cationic polymers that are used to deliver microparticles that efficiently guard CRISPR-Cas9 technology are positively charged. This is a big issue because, as positive and negatively charged particles attract each other, the cationic polymers will naturally adhere to the mucus, preventing penetration of the mucus layer. PEG coating effectively switches the microparticle charge to neutral.
Note, recently scientists have identified another type of cationic polymer molecule called spermine that has been shown to have higher efficiency to penetrate the mucus than PEI
Higher transfection rates (Yuan & Li, 2017)
Better biocompatibility (Yuan & Li, 2017)
Previous studies have shown that spermine-based polymers can reach target genes with relatively low toxicity (Yuan & Li, 2017)
Once the aerosol particles pass through the mucus layer, they also must bypass the periciliary layer, also called the PCL before reaching the cells (Chow et al., 2020). It comprises of cellularly-tethered mucins in a mesh-like arrangement, sharing resemblance to the mucus layer (Chow et al., 2020). Along this layer within the airways there are also pulmonary surfactants that are secreted by the epithelial cells in the alveolar region of the lungs (Chow et al., 2020).
Pulmonary surfactants are a combination of various proteins and lipids, which are negatively charged. Their negative charge is important because it provides another issue for the particle to bypass: a second negative layer will hinder the movement of a positively charged particle. Therefore, extra attention should be paid when administering the neutralizing PEG coating. The anionic sections of the surfactants (phospholipids, cholesterols, and surfactant proteins) can cause predisposition of the aerosol particles.
Figure 4: Movement of nanoparticles (NPs) through various blockages. Retrieved from ‘Comparative study of mucoadhesive and mucus-penetrative nanoparticles based on phospholipid complex to overcome the mucus barrier for inhaled delivery of baicalein, 2020.’
Mucus-penetrating particles are rapidly cleared through the mucus layer, but particle movement through the periciliary layer is considerably slower. This is generally due to the fact that the mucus layer is usually under 5μm thick, while the PCL is 7+μm thick (Fahy & Dickey, 2014). Since the layers are very similar in composition, this added width would hinder the movement in particles specifically through the PCL.
2.1 Outcome: Airway Surface Liquid
For microparticles to efficiently penetrate the mucus and periciliary layer:
Polymeric gene carriers should be used, specifically cationic polymers.
The polyplex with the composition of the PEI-MPP should consist of 25% pure PEI and 75% PEG5k –PEI should be used.
Additional research is required to assess if spermine-based cationic polymers can replace the PEI-MPP for a higher transfection efficiency.
The airways of CF patients are often inflamed and infectious, as the mucus is a breeding ground for bacteria. This creates a more toxic, and turbulent environment for the particles to move through. This means that, on average, the particles will not travel as far as usual, their probability of making it to the epithelial cells in the bronchi is slim. Drugs such as ibuprofen and corticosteroids have been used to reduce lung inflammation. These measures should be administered right before aerosol inhalation to reduce overall airway inflammation and to increase ease of access to the bronchi.
3.0 Distribution to Bronchi Epithelium
This is the destination of the aerosol’s journey. When the aerosol particles reach the epithelium, they undergo mediated endocytosis. Endocytosis is a form of active cell transportation that delivers molecules, partial cells, and whole cells into a single cell (Lumen Learning, n.d.).
Figure 5: Movement of polyplex movement through a cell
Here are the complete steps for a successful polyplex internalization/nucleus transportation:
The endocytosis process consists of two main steps. First, the cell membrane ‘invaginates’ or forms a type of particle-like barrier around the incoming material (Alberts, 2002). Then the cell is brought into the cell in an intracellular vesicle created from the membrane (Alberts, 2002).
After endocytosis, the DNA plasmid needs to escape the intracellular vesicle before entering the nucleus, or the CRISPR-editing technology will not reach the cell’s genes at all.
PEI is a titratable amine-containing polycations (types of polymers that have multiple positive charges and are made up of amines - nitrogen-based derivatives from ammonia - that can undergo titration).
Titratable amine-containing polycations may proceed through endosomal escape (a.k.a. exiting the vesicle) through the Proton-Sponge effect.
The Proton-Sponge Effect is a process in which the presence of a weak, basic molecule will cause the endosome (vesicle barrier) to burst (Freeman et al., 2013). This works as the polyamines, while in the endosome, will attract free protons. These protons will accumulate within the endosome (as protons cannot escape the endosomal barrier through diffusion) until the membrane potential is past equilibrium. This will cause a reaction: membrane equilibrium is affected by chloride diffusion. Thus, chloride will automatically diffuse into the endosome barrier. This will cause the endosome to swell and expand, eventually leading to explosion (rupture) and the release of the DNA plasmid.
Figure 6: Display of Proton-Sponge Effect in an endosome.
The release of genetic material can occur either in parallel with endosomal escape or by dissociation triggered by a cytoplasm-induced reaction (polyplexes have been inferred to dissociate when in contact with cellular cytoplasm, but scientists have not pinpointed what mechanism causes this).
Further, there are two ways DNA plasmids can enter the nucleus.
When bronchial epithelium cells undergo mitosis (airway epithelial cells turnover slowly at < 1% per 24 h, unless affected by epithelial cell injury - which results in faster cell turnover due to proliferation) the envelope is temporarily separated. As the turnover rate is so minimal, it is not very time efficient.
A faster approach is nuclear membrane penetration through the nuclear pore complex. The nuclear pore complex is an aqueous channel (water-based solvent) through which proteins, macromolecules, and ribonuclear proteins can pass through (Bai et al., 2017). The pores are 0.06μm - the microparticles designed so far would not be able to naturally penetrate the NPC.
For the ‘naked plasmid’ to enter the NPC they need to be paired with a Nuclear Localization Sequence, also known as an NLS. Research from Dr. Collas and Aleström have shown that copies of plasmid DNA that were complexed to NLS peptides had effectively increased nuclear uptake (nuclear internalization). This testing was done of germline nucleuses, but the same methods can be applied to somatic (body) cells.
3.1 Outcome: Distribution of Bronchi Epithelium
For DNA plasmids containing the CRISPR-Cas9 molecule, the route/methods to the bronchial epithelium nucleus goes as follows:
The polyplex undergoes endocytosis.
While inside the intracellular vesicle, the polyplex will undergo endosomal escape through the proton-sponge effect.
The now naked DNA plasmid will enter the nuclear pore complex using NLS peptides.
4.0 CRIPR-Cas9 Integration
Once the DNA plasmid containing the Cas9 enzyme and guide RNA (gRNA) enters the nucleus, there is a general process in editing the mutated gene. The gRNa is an RNA strand that matches with the mutated DNA, so it travels along the specific gene that contains the mutated gene.
For cystic fibrosis, the CFTR gene is positioned on chromosome 7. Once the gRNA matches with the section of DNA that is supposed to be edited, the Cas9 enzyme cuts the mutation out. For the F508del mutation, since there is loss of nucleotides coding for an amino acid, the CRISPR-Cas9 molecule would introduce a repair template. This repair template would have a corrected version of the F508del code, with the amino acid. Now the DNA would carry the correct sequence.
Figure 7: Explanation of CRISPR implementation through gene editing.
5.0 Effects of Time Progression on Inhalable Treatment
Time plays a great factor in the type of treatment cystic fibrosis patients must receive; time has direct correction to the state of the affected lung. Pediatric cystic fibrosis lung biopsies often show evidence of neutrophilic airway inflammation and mucus plugging leading to lung damage through diffuse bronchiectasis, and lung parenchyma deterioration in adult populations as time progresses (Balazs & Mall, 2019).
Neutrophils are a type of circulating leukocyte and make up around 70% of white blood cells. Thus, they play a big role in the human body’s immune system and responses. When the body identifies and infects, neutrophils are responsible for travelling to the infected site and use their specific mechanisms to battle against invading pathogens (Jasper et al., 2019). In the case of cystic fibrosis, these specific neutrophils can release MPO and NE, types of immune proteases that fight infection, but at extreme doses. This translates into tissue degradation and continuation of inflammation. Innovative treatment agents targeting airway smooth muscle hypertrophy as well as airway remodelling have been used to treat inflammatory neutrophils (Syabbalo, 2020). Some examples of studied agent/treatment options are phosphodiesterase 4 inhibitors, macrolide antibiotics and bronchial thermoplasty (Syabbalo, 2020).
Furthermore, there are types of neutrophils that can hold, and release DNA fibers decorated with anti‐microbial proteins. These are called neutrophil extracellular traps (NETs). NETs are associated with certain molecules who are known to be able to kill epithelial cells, which have been found in CF-affected lungs. There has been recent progress pertaining to the attack of pulmonary NETs. This includes using recombinant human DNase therapy and chloroquine/PAD4 inhibitor drug treatment for DNA disintegration, and anti-histone antibodies/various protease inhibitors for NET protein neutralization (Porto & Stein, 2016).
Mucus plugging is known as one of the earliest signs of cystic fibrosis in pediatric patients. Mucus plugs are inspissated formations of mucus within airways. The treatment of pediatric patients with BiPAP treatment paired with a bronchoscopy has been tested, resulting with positive results (Elidottir, 2018).
Diffuse bronchiectasis is the destruction and/or dilation of larger-shaped bronchi throughout many areas of the lung (as opposed to focal bronchiectasis which only affects few and specific regions of the lung) (Merck Manual, n.d.). It can easily be treated using antibiotics (oral or IV), mucus thinning medication treated using a nebulizer, or the use of airway clearance devices that are used to disintegrate mucus buildups (IPVs, PEPs).
Lung parenchyma refers to the region of the lung that is involved in the exchange of gases (predominantly oxygen and carbon dioxide), the primary pulmonary structure being the alveolus (Pinkerton et al., 2015). In terms of lung parenchyma deterioration, it is a process that takes place over periods of time and coincides with lung function and structure deterioration. There have been many restorative therapies studied to try and reinstate the lung to its healthy state, but no conclusive treatment is available.
The word ‘polymicrobial infections’ refers to infections that involve multiple infectious agents and are referred to as complex, complicated, mixed, dual, secondary, synergistic, concurrent, polymicrobial, coinfections (Haverkos, 2003). The organisms affecting CF children historically are H. influenzae and S. aureus, while adults are predominantly affected by P. aeruginosa or Burkholderia cepacia (Filkins & O’Toole, 2015). Treatments for these few, and basic bacterial organisms include use of antibiotics (H. influenzae: amoxicillin/clavulanate, azithromycin, cephalosporins, fluoroquinolones, and clarithromycin; S. aureus: linezolid (both IV and oral) and IV vancomycin; P. aeruginosa: nebulised colistin, tobramycin and gentamicin; Burkholderia cepacia: systemic antimicrobials such as ceftazidime and meropenem) (Merck Manual, n.d.; Esposito et al., 2019; Banerjee & Stableforth, 2000; Pegues, 1996).
Any given cystic fibrosis patient may suffer from combinations of the above complications and many more individually based, often related to age, genetics, and other biological variables.
For these issues, scientists must apply the theorized inhalable method assisted by commonplace CF treatments in a patient-specific manner, whether that be through preparatory antibiotics or the use of a BiPAP device prior to the inhalation of the CRISPR polyplex. This would help, as the chances of airways being less constricted are higher. Bigger airways result in particles making an easier journey to the target cell. Overall particle deposition would decrease, making the chances of reaching the cell higher. BiPaP devices administer a positive breathing pressure and are commonly used as breathing assist devices.
DISCUSSION & FUTURE STEPS
As it always is with genetic therapies, one should understand that the research must encompass not only biologically successful and performance-based approaches, but also thoroughly investigate from a patient-centered point of view. The patient’s experience is one of the most important variables at hand, especially in a disease as lifelong as cystic fibrosis. Patients already are put through dozens of antibiotics and are put through extremely heavy rotations of, in some cases, extreme doses of medication. This research and the therapy proposed can be improved in many different aspects. This includes the use of a less toxic, caustic cationic polymer, antibiotics before and after inhalation, differences in therapy processes pertinent to age and disease state, and the development of a more efficient CRISPR-based carrier.
Phosphodiesterase 4 inhibitors, also known as PDE4 blockades, were tested in vitro (healthy human neutrophil samples) and in vivo (mouse models). In vitro, PDE4 managed to stop endotoxin-induced NET production while maintaining overall cell stability and structural integrity (Totani et al., 2021). When tested in vivo, the mouse models showed that alongside in vitro results, the amount of ‘free DNA’ found in the bronchoalveolar lavage fluid had significantly decreased (Totani et al., 2021).
Macrolide antibiotics are a family of compound chemicals that are frequently used in respiratory disease medicine, due to easy oral administration, incredible tissue penetration, and widespread efficiency against pathogens within the lung (Spagnolo, 2013). Erythromycin, a type of macrolide, taken at 600 mg daily, proved that it could reduce the number of neutrophils in the bronchoalveolar lavage fluid of patients (Spagnolo, 2013).
Recombinant human DNase therapies have been explored in relation to NETs found in sputum collected from extreme Covid-19 case patients. DNase therapies combined with albuterol showed promising results: increased oxygenation and decreased lung opacity (Fisher et al., 2021).
Anti-histone antibodies have also been used against NET protein neutralization. By removing the cytotoxic histones from NETs, the the NET’s structure is effectively destabilized (O’Meara et al., 2020). Polysaccharides, like heparin and polysialic acid are a type of polymeric carbohydrate, which can produce O-sulfated small polyanions (SPAs). SPAs can inhibit histone toxicity (O’Meara et al., 2020).
BiPAP treatment paired with a bronchoscopy has been successfully used as an airway clearance technique. For severe cases, this technique can clinically stabilize a patient. Fourteen out of sixty-five patients testested had used BiPaP therapy against mucus plugging, and ten out of those fourteen had also endured a bronchoscopy. The combined treatments were taken well by all patients and successful in nine (Elidottir, 2018). This is an extreme method, and may not be as patient-friendly as others. It required a significant amount of exposure and lung remodelling.
Mucus thinning medication, such as hypertonic saline, can be given in concentrations, increasing the sodium percentage at every increment. The salt plays the role of the CFTR protein, attracting water outside of the cells, making the mucus thinner, and easier to cough (Cystic Fibrosis Foundation, n. d.) Long-term treatment of 7% hypertonic saline, four times a day, resulted in a ≥8 hours increase in 1-hour rates of mucosal clearance (Donaldson et al., 2006). This technique is used frequently, considering its high success rates.
CONCLUSIONs
Moving forward, I would like to spend more time on each of the biological barriers I have outlined and improve them individually to the best of my ability. Specifically, I would invest time into researching the use of viral molecules, upcoming genetic barcoding technology and nuclear proteins to better ‘lead’ microparticles to their target genes, resulting in better editing efficiency. Apposite to the therapy itself, I would delve deeper into the variables affecting the number of times the therapy has to be applied, and how we may effectively decrease that number.
Even though CF is a multi-organ disease, improving respiratory related health issues by genetic intervention in lungs could result in a significant improvement in the patient’s quality of life and decreased mortality. With the recent development of powerful tools to manipulate the genome, new perspectives are opened in gene therapy to restore the correct CFTR expression. This research presented here involves the use of an inhalable aerosoland its delivery strategies as a means of CF therapy through gene correction of CFTR mutations. Genome engineering and modulation is at the forefront of medicine, not simply through novel approaches, but also for the development of experimental models critical to developing effective translational medicine. Despite marked progress as of recent, there is much to be done in terms of tools of nucleic acid delivery and genome editing molecules in-vivo. This report proposes an original blueprint of an inhalation-based method to CRISPR-driven molecular genetic engineering for the treatment of CF.
Figure 8
ACKNOWLEDGEMENTS
I would like to thank geneticists Dr. Rachel Wevrick from the University of Alberta and Dr. Johanna Rommens from the University of Toronto (who was part of the original team who discovered the CFTR gene) for mentoring/advising me while I conducted the research, my parents for their incomparable support, my friends, and peers for their read-overs, and last but not least, Ms. Wong and Mrs. Joblinski, my science teachers for their continued motivation.
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about the author
Maya Achuthan
Maya Achuthan is a Grade 10 student at St. Michaels University School in Victoria, BC. She is fascinated by the relationship between genomics and the inner workings of the human brain and is eager to learn more about the increasing use of software, AI, and biotechnology in the field of medical genetics. Maya is the Co-founder and CEO of Helpi5, an app that supports companies in creating “in-app” help content, improving customer experience, and is an Outreach Ambassador for the STEM Fellowship Organization. In her spare time, she enjoys biking, rowing, badminton, and reading tons of books.