Novel Gene Therapies for Cystic Fibrosis

Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive, life-limiting disease resulting from gene mutations that encode the cystic fibrosis transmembrane conductance regulator (CFTR). This gene is comprised of 27 exons and is located on chromosome 7. CFTR is a cAMP-regulated chloride channel located on the apical membrane of exocrine epithelial cells. CFTR is involved in regulating the epithelial sodium channel and bicarbonate transport. There are many different types of CFTR mutations that result in CF; lack of production; failure to reach the site of action; defects in gating; conductance; abnormally low channel numbers; and decreased half-life. The significant effect of these mutations is experienced in the respiratory, gastrointestinal, and reproductive tracts. Thick, viscous secretions often obstruct these tracts.  

Existing therapies for CF mainly focus on mechanical clearance of airway secretions and treatments for infections. In recent decades, improvements in airway clearance technology and nutrition have significantly improved life expectancy and quality of life. However, these are palliative measures fraught with difficulties, including bacterial resistance and time-consuming nature. Life expectancy is 44 years old without a lung transplant, and 90% of patients die from advanced lung diseases.  

Viral gene therapy in CF

Gene therapy is theoretically well-suited for CF; CF is a single-gene disorder; heterozygotes are phenotypically normal; airways are easily accessible in theory; lungs are normal at birth, indicating a potential therapeutic window. However, in practice, airways are challenging targets for gene therapy. You find the mucociliary escalator in the lungs, which has evolved to keep foreign particles out. In healthy lungs, mucus is produced and aids in normal ciliary function and inhibits gene transfer, and in CF, mucus secretion is excessive and highly viscous. In addition, the human immune response can also be troublesome. The viral gene transfer agents, recognition of viral coat proteins, and production of neutralising antibodies can lead to problems with re-administration. If non-viral gene transfer agents are used, the DNA plasmids are rich in unmethylated CpG dinucleotides; this tends to be recognised as foreign by the immune system triggering an immune response.  

Adenovirus based gene therapy

Various pharmaceutical companies are investigating many gene therapy-based treatments. A year after the CFTR gene was found to be the culprit behind CF, two separate groups of researchers demonstrated that it was possible to introduce the gene to ex vivo cells using a viral vector, triggering the production of a functional CFTR protein. Adenoviruses (AD) were initially very popular; they usually cause the common cold and other respiratory tract infections, although researchers use non-pathological versions that have been engineered to carry a function CFTR gene. The gene is introduced into the person's cells; however, the sequence doesn't integrate into the patient's DNA. However, clinical trials using this method had lacklustre results. As this research developed, scientists began to worry that repeated administration of AD-based treatments could trigger an immune response and neutralise the virus. Sadly, in 1999 an 18-year-old Jesse Gelsinger was enrolled in a clinical trial for AD-based therapy for ornithine transcarbamylase, and after four days, he died from a massive cytokine storm.  

ADs have many advantages as a gene therapy: high transduction efficiency; epichromosomal persistence in the host cell; broad tropism for different tissues; availability for scalable production systems; large packaging capacities of about 36 kilobases (kb).

What are adenoviruses

ADs are non-enveloped viruses known to infect the upper respiratory tract, brain, and bladder. It possesses an icosahedral protein capsid to accommodate a 26-to-45-kb linear, double-stranded DNA genome. ADs are flanked on either side by hairpin-like inverted terminal repeats (ITRs), these act as self-priming structures that promote primase-independent DNA replication. Infection of AD begins with the interaction between cell surface-localised coxsackievirus-AD receptor (CAR) and the distal domain of virus capsid fibre. There are a variety of other receptors for AD entry; these include CD46, DSG2, and sialic acid. These interactions are followed by endocytosis, mediated by interactions between the tripeptide Arg-Gly-Asp motif in the penton base and the alpha V integrins on the host cell surface. Post endocytosis, the capsid is disassembled, and V and VI proteins facilitate endosomal release. The viral DNA then enters the nucleus through the nuclear envelope pore complex.    

What is a cytokine storm?

Cytokine storms or cytokine release syndrome are life-threatening systemic inflammatory syndromes that show elevated circulating cytokine levels and immune cell hyperactivation. Cytokine storms have been thought to heavily contribute to the lethality of the 1918-1919 influence pandemic. Various disorders have been thought to cause cytokine storms, such as sepsis, primary and secondary hemophagocytic lymphohistiocytosis (HLM), autoinflammatory conditions, and coronavirus disease 2019.

Adeno-associated viruses (AVVs) based gene therapy

After the tragic death of Jesse Gelsinger, researchers partially abandoned adenoviruses and switched to adeno-associated viruses (AAVs). Genetic engineering of recombinant AAV (rAAV) has demonstrated that packaging of transgenes could be achieved by gutting the genome, retaining the ITR elements, and replacing it with promoter and gene of interest. When AAVs were first studied in humans to deliver the CFTR gene in combination with the AAV2 capsid into a CF patient. AAVs were considered to trigger less of an immune response than adenoviruses. A group did a trial in 1999 at Stanford University School of Medicine, in which ten patients with CF were given gene therapy with an AAV vector. These trial and subsequent ones showed that AAV vectors did not produce a major immune response, but neither did it show any massive improvement in symptoms. A significant consideration of AAV vector design is that the wild-type genome is about 4.7kb in size; as a result, the vector design is limited to a kilobase capacity; components required for proper expression need to be truncated to fit into the capsid. AAV is still being pursued by CF treatment today. Companies have engineered AAVs to be more infectious, enabling them to reach more cells in the lungs; the structural complexity of the lungs, with smaller and smaller branches, makes administering therapies deeply into the lungs a problem.

There are some limitations to AAV, the size. The AAV vector has a size limit of 4.7 kilobases, and the CFTR gene is 4.6 kilobases in length; there is no remaining space for incorporating promoters that would boost the production of the CFTR protein levels. Although, some companies have engineered a functional CFTR gene that's shorter, allowing the AAV vector to contain the gene and promoters.

Lentivirus gene-based therapy

Lentiviruses are members of the retroviridae family. Lentiviral particles encapsulate two sense-stranded RNAs that are bound by nucleocapsid proteins. The particle also contains reverse transcriptase, integrase, and protease proteins. Infectious lentiviral particles enter cells through interactions between glycoproteins and cell receptors. Successful binding prompts fusion between the viral particle lipid bilayer and cell membrane, resulting in the release of genetic cargo into the cytoplasm. The integration of lentiviral DNA into host DNA is non-random in manner, displaying a preference for transcriptionally active sites; this might mean that it can be administered less often than other forms of therapy.

Lentiviruses have many features that are amenable to transgene delivery for therapeutic purposes. Lentiviruses are integrating vectors that permit long-term transgene expression. They have a larger packaging capacity, the AAV of up to 9 kilobases. Lentiviruses vectors have been shown to express multiple genes from a single vector. Studies have also shown that lentiviruses are less likely to be neutralised and destroyed by the immune system than AAVs. The UK cystic fibrosis gene therapy consortium, combined with Boehringer Ingelheim, a German pharmaceutical company, is pursuing a lentivirus-based gene therapeutic for CF; this is at the preclinical stage.        

Non-viral gene therapy

There have been studies investigating non-viral methods to deliver gene-based therapy. The first approach is to inject the CFTR gene directly surrounded by lipid molecules to shield the genetic material from degradation. In 2015, the UK Cystic Fibrosis Gene Therapy Consortium put a liposome-based treatment through clinical trials, reaching phase IIb; however, this approach never greatly improved lung function. A second approach being investigated is to deliver RNA, encoding for the correct CFTR protein production so it can serve as a template for protein production. A third approach is to engineer transfer RNA (tRNA) molecules that trick the cellular machinery into producing correct CFTR. This method is being pursued because some CFTR mutations result in the premature recruitment of tRNA molecules that signal the fault of CFTR production. tRNAs are short sequences that transport amino acids to the cellular machinery.

Some drug developers are working on read-through approaches which encourage cells to ignore premature stop signals in specific CFTR mutations. Molecules have been developed to trick the ribosome into assembling functional CFTR proteins. This method has been brought to phase II trials by Eloxx pharmaceutical, but trials were halted due to the COVID pandemic.

CRISPER-Cas9 is a method that directly edits the patients' cells, resulting in cells producing a functional version of CFTR proteins after treatment. CRISPER technology has shown to have promising results with organoids. Organoids are clusters of patients' cells grown to show some characteristics of the organs in question. However, it must reach the lungs' stem cells for CRISPER and lentivirus technology to provide an actual cure. Although, there are concerns that CRISPER therapies might edit in the wrong place resulting in cancerous mutations.