Introduction

Overview of Cystic Fibrosis (CF)

Cystic Fibrosis is a condition caused by a genetic mutation in the gene CFTR. 

A genetic mutation is a random change in DNA sequence. The gene CFTR codes for a protein that allows salt to move from inside of the cell to outside, and subsequently water will also move out to form mucus with a thin texture. Mucus is important for trapping dirt and bacteria, and they would get swept down the airway so the pathogenic substances can be destroyed. The mucus also lines important passageways in the body to protect and moisten them. 

However, in CF the mutated gene causes an unfunctional protein to be made, so salt can not move out and ultimately, the lack of water in the mucus means that the mucus is very thick and sticky, causing the passageways in the body, especially the respiratory and digestive systems, to cause blockage and inflammation. 

Therefore some of the major symptoms of CF include:

• Chest infection
• Dry coughs and wheezing
• Diarrhoea and constipation

CF is an inherited disorder. As humans, we inherit two versions of a gene from our parents, one from each. In order to be positive for CF, the child needs one copy of the faulty CFTR gene from each of the parent, if the child only has one copy, then they are said to be “carriers” of the CF. 

Unfortunately there is no cure for the condition, however there are a few treatment approaches that can manage the symptoms:

• Antibiotic to prevent chest infection
• Medications that thin down mucus
• Bronchodilators to widen airways
• Physical exercise
• Lung transplant

Concept of Gene Therapy

Gene therapy is a form of treatment that alters a faltered gene inside cells to treat genetic disorder and cancer.

Regarding CF, a correct version of the CFTR gene will be implemented into the patients’ body to allow a functional protein to be made. The gene therapy mainly targets cells that are affected by CF, ie. epithelial cells that line the respiratory system. The current gene therapy method called somatic therapy does not target sex cells; therefore, the patients’ offspring may still inherit CF.

Gene therapy still exist primarily in a research setting, and faces some ethical issues, nevertheless using gene therpay to treat cystic fibrosis has shown some promising result. 

Mechanism of Gene Therapy for CF

The objective of gene therapy for CF is to place a correct copy of CFTR gene into the body, so cells can synthesise functional CFTR protein. Depending on how the gene is delivered, the synthesis of CFTR protein can be permanent or temporary.

Methods of delivery

Viral vectors (e.g., adenoviruses, lentiviruses)

Viruses consists of DNA strand in coated in a protein shell called a capsid; there are thousands of different types of viruses and each type of virus can only invade a few types of human cells. Scientist have utilised this property of virus by replacing the harmful genetic material inside the virus and replaced with a corrected gene for gene therapy so viruses can “invade” lung cells to allow cells to take up the corrected CFTR gene.

Lentivirus is quite effective in delivering CFTR gene to epithelial cells lining the airways of the lungs. They bind to the specific receptor on the surface of the lung cells and fuse with the membrane to allow the protein coat containing the corrected gene, enter the target shell. 

Non-viral vectors (e.g., lipid nanoparticles, electroporation)

Non-viral vectors mean that the therapeutic gene is not delivered to target cells using a virus. Genetic materials can be delivered using physical method or chemical carriers. 

The physical delivery method usually transports genetic material without anything protecting it. One example of this is electroportation, which involves applying heat-shock to the cell-membrane which will be permeable to the genetic material for a short time to allow it to enter the cells.¹

Chemical carriers refer to vectors such as lipid nanoparticles. Lipid nanoparticles have a positively-charged section in the structure, and they interact with the negative part of the DNA in the therapeutic gene because opposite charges attract. The positive charge of the molecule also allows them to interact with the cell-surface protein to allow the particle to enter the cells where functional CFTR proteins are made. 

There are advantages of non-viral over viral vectors. Since the lipid particles are bigger than viral particles, the method allows bigger therapeutic genes to be ported into the cells. However, it is less effective in delivering the gene into the nucleus of the cells, therefore the effect of gene therapy is more temporary. 

Types of Gene Therapy Approaches

Gene Augmentation Therapy

Gene augmentation fully utilises the vectors, both non-viral and viral types, as the corrected gene needs to be delivered to lung cells. The functional CFTR protein can be synthesised when the corrected gene is in the cells. The delivery of the gene through non-viral vector has improved the successful transport of chloride ions in salt by 20%.¹ Though genes delivered by viral vectors do demonstrate longer lasting effects through the expression of functional protein than non-viral vectors, current studies still struggle to demonstrate any evidence of gene augmentation therapy fully restoring the functions of the lungs.

Gene Editing- CRISPR/Cas9

By using CRISPR technology, scientists are hoping to correct the mutation in the CFTR gene in patients. CRISPR stands for clustered regularly interspaced short palindromic repeats. It consists of two components: the Cas9 protein, which can cut DNA, and a guide RNA, which can recognize the sequence of DNA to be edited. To use CRISPR-Cas9, scientists first identify the sequence of the human genome that's causing a health problem. They then create a specific guide RNA to recognize that particular stretch of A's, T's, G's, and C's in the DNA. The guide RNA is attached to the DNA-cutting enzyme Cas9, and this complex is introduced to the target cells. It locates the target letter sequence and cuts the DNA at that point. Scientists can then edit the existing genome by modifying, deleting, or inserting new sequences, effectively making CRISPR-Cas9 a cut-and-paste tool for DNA editing.

RNA-based Therapies

RNA-based therapies target types of CFTR mutations that are a result of incorrect splicing of mRNA. 

A complete DNA that codes for CFTR stays in the nucleus of a cell and is unable to exit the nucleus to the cytoplasm where the actual CFTR protein is made because DNA is too big. Therefore, DNA is first made into mRNA which is much smaller and shorter than DNA in the nucleus, then mRNA exits the nucleus to the cytoplasm for the synthesis of CFTR protein. During the conversion of DNA to mRNA, the “unuseful” sections (introns) of DNA that do not code for CFTR proteins are removed (spliced), and the “useful” sections (introns) are joined back together. mRNAs that have not yet had the exons removed are called pre-mRNA. So, in RNA based therapies, molecules called antisense oligonucleotides (ASOs) modify pre-mRNA to ensure correct splicing is performed to ensure correct mRNAs are made to allow correct CFTR proteins to be made.³

Gene Silencing

As the name suggests, this method aims to silence the mutated CFTR genes to reduce the synthesis of faulty CFTR genes. This is achieved by using ASOs, also used in RNA-based therapy and siRNA which works similarly to ASOs. They bind to mRNA made from mutated CFTR DNA, and the complex will either get destroyed by another protein called RISC or destroyed by the cell’s own mechanism. 

Current Challenges and Limitations

Immune response to viral vectors.

There are some significant challenges in developing a vector that can successfully deliver the genetic material to lung tissue, with immune response being the major one. The human body is very good at identifying things that are not part of its system, so when the body detects vectors as foreign molecules, the immune system tries to destroy them, preventing the genetic materials from reaching the target. 

Difficulty in targeting lung cells due to mucus buildup.

In both healthy and CF individuals, mucus that lines the airway is present to trap any potentially “harmful” substance. Especially for non-viral vectors, the genes coated in lipid coat are usually physically inhaled for gene delivery, so the mucus layer will trap the vectors making it more difficult for genes to reach targets. 

Summary

In conclusion, although gene therapy for cystic fibrosis (CF) is still at a quite early stage of clinical research, it offers a promising approach to address the underlying genetic mutation responsible for the disease. Gene therapy attempts to fix the faulty CFTR gene, by restoring normal protein activity. Non-viral techniques like lipid nanoparticles offer safer alternatives with some efficacious limits, while viral vectors like lentiviruses and adenoviruses have demonstrated promise in delivering the repaired gene. Technological developments in gene-editing instruments like as CRISPR/Cas9 and RNA-based therapeutics have the potential to enhance treatment approaches, target certain mutations, and yield better results. Significant obstacles still need to be overcome, such as immunological reactions to viral vectors and the difficulty of accurately targeting lung cells because of mucus accumulation. To overcome these obstacles and provide safe, efficient gene therapies that will greatly enhance the quality of life for CF patients and possibly provide a long-term cure for the illness, more research is necessary.