How A Gene Encoding A Therapeutic Protein Could Be Cloned Into A Vector To Allow Expression In Gene Therapy

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How A Gene Encoding A Therapeutic Protein Could Be Cloned Into A Vector To Allow Expression In Gene Therapy

A gene is a nucleotide sequence which dictates the synthesis of a particular RNA or protein molecule. Their control over the produced proteins govern both phenotypical and genetic traits, including susceptibility to diseases like Cystic Fibrosis. Driving gene expression is Central Dogma, a two-step process in which DNA is converted to an intermediate RNA (mRNA) through transcription, then from mRNA to protein through translation. Virtually all living and acellular beings abide to Central Dogma bar the Retrovirus family and viruses such as Hepatitis B Virus (HBV) (Madigan et al., 2012). These specialised viruses are capable of producing an enzyme called Reverse-Transcriptase (RT) and this converts their single-stranded RNA molecules into double-stranded complementary DNA (cDNA), allowing them to integrate into the hosts genome in a process termed lysogeny (Madigan et al., 2012).

As technology and the understanding of genomics thrive, diseases once thought incurable are being corrected through a revolutionary process called Gene Therapy. Gene therapy is where a gene is delivered to an individual in order to correct a genetic disease caused by a faulty version of the replacement gene. There are two versions of gene therapy, somatic and germ-line. Somatic is limited to the lifetime of the individual while germ-line prevents the defective gene from being passed onto future descendants, the latter being considered highly unethical and currently banned due to its ability of affecting subsequent generations. To generate new and functional copies of defective genes, DNA cloning vectors are essential. A vector is a virus or DNA which can be utilised to carry and replicate other pieces of DNA. (Thieman and Palladino, 2009). Ever since the construction of pSC101, the number of DNA vectors has exponentially increased with plasmids being the most popular cloning vectors for being easily transformable and isolatable from their host bacterium. Unfortunately, plasmids have their limitations just like all the other vectors in Table 1. Thus, to undergo Gene therapy, it requires several vectors (Thieman and Palladino, 2009).

Previously, genes for deficiencies such as insulin deficiency in diabetes mellitus patients were taken from animals, but this carried risk of zoonotic diseases. Thus, the understanding of bacterial genomics was ground-breaking. Certain species of bacteria are competent, meaning that they are willingly able to uptake naked DNA strands which shall give them a selective advantage (Madigan et al., 2012). Escherichia coli (E. coli) is the most studied model bacterium and is favoured for majority of lab work, particularly in recombinant DNA cloning practises. For transformation to occur, the plasmid DNA vector needs to contain several characteristics, including a Multiple Cloning Site (MCS), Antibacterial resistance gene, Origin of replication (Ori) and selectable marker genes.

The antibacterial resistance gene is highly important as it indicates which colonies have been transformed and house the target gene. For example, if the growth medium was embedded with Ampicillin, the plasmid DNA would be constructed to contain the AMPR gene so that only competent bacterium cells would be present on the agar plates (Thieman and Palladino, 2009). The colonies are grown until stationary phase (optimum stage) and one colony is selected and grown in a selective liquid culture medium. The grown E. coli cells are centrifuged into a pellet which is subjected to the PureLink Quick Plasmid Miniprep protocol. The first step is resuspension. Resuspension buffer uses EDTA, a chemical which chelates the divalent cations Mg2+ and Ca2+, Dnase cofactors that degrade plasmid DNA. However, with EDTA present, the cofactors cannot initiate Dnase activity and thereby protects the plasmid DNA. Rnase is also present and gives a purer DNA sample by degrading RNA present in the cell. Next, lysis buffer, which contains Sodium Hydroxide (NaOH) and Sodium Dodecyl Sulphate (SDS), is added. NaOH denatures any proteins and SDS lyses the cells membranes. Potassium acetate, an acidic salt, is added to neutralise the basic effect of NaOH and the degraded proteins shall precipitate and are easily removed by centrifugation, leaving the supernatant containing the plasmid. The supernatant is transferred to a spin column where the plasmid DNA binds. Any impurities present are washed away using an ethanol wash, leaving the plasmid DNA bound to column while the flow-through accumulates in the wash column before being discarded.

After two centrifugations, the column is placed into a recovery tube and centrifuged so that the plasmid DNA is eluted by Tris-HCl from the column into the recovery tube. Its critical that the recovered plasmid DNA is moderately concentrated and pure as otherwise the remaining stages are affected. Both of these qualities are tested by use of gel electrophoresis. By using gel electrophoresis, the DNA bands which appear shall not only indicate the band size but also its purity as, the brighter it is, the purer it is. If no band appears, it indicates that the bacterium wasnt competent or that the protocol aforementioned wasnt strictly followed. To quantify the results, one could use spectrophotometry or fluorometry to determine the concentration of plasmid DNA. The next stage involves restriction digestion. This stage is crucial because, to ensure efficient transformation into the destination vector, specific restriction enzymes are chosen to act as surgical scissors, cutting in-between specific nucleotides in the DNA sequence and generating sticky ends. The term sticky ends refers to both ends having an uneven distribution of nucleotides on either end. The samples are then loaded onto a gel electrophoresis with the purpose being to calculate how much insert needs to be added during ligation. The bands are viewed under UV light, photographed and cut out quickly to avoid risk of thymine-dimerization. The gel slices are dissolved and purified, and the contents mixed together, allowing the linearized vector to ligate into the destination vector which has had an exact same cut in its own DNA sequence, thereby ensuring that the donor vectors DNA adherently binds in the correct conformational orientation.

Once ligated, the same transformation process from earlier is used. Only transformed bacteria with the destination vector will grow in colonies on the plates. To confirm the ligation was successful, random colonies will be selected for Polymerase Chain Reaction (PCR).

(1) Double-stranded DNA is denatured at 950C, leaving single-stranded DNA molecules. (2) Temperature is lowered between 40-600C depending on primers. The primers anneal to the strands, flanking the site of interest. (3) The temperature is raised to 720C where extension occurs. Taq DNA Polymerase synthesises new DNA strands, creating two new double-stranded DNA molecules. The steps repeat for another 30 cycles with the number of target DNA being generated exponentially increasing.

Today, gene therapy is necessary to treat genetic disorders, including dominant and recessive genetic disorders. Table 2 showcases several medical conditions currently being studied and at what trial stage. Gene therapy can take one of two approaches; viral and non-viral. Viral delivery is more specific to disorders affecting the central nervous system (CNS) as the majority of other available therapeutic approaches struggle to breach the blood-brain barrier and those that do fail to have effects on the target cells (Qu et al., 2019). As for non-viral delivery, methods include using a Gene-gun, a direct contact methodology in which a high-velocity injection of DNA-coated fragments transform the cells, direct injection of the vector, and liposomes, small spheres that contain DNA with artificial membranes which can fuse with target cells membranes and release their contents (Krebs et al., 2014). Liposomes theoretically fuse with any cell type, but their affect is only short-lived, and symptoms start to reappear. Direct injection of the vector has proven to be a worthwhile methodology, especially in the treatment of lung cancer. By mixing a functional p53 gene into an injectable drug, the gene can bypass the immune system reaction, a frequently occurrent problem with other gene therapy techniques, and suppress cell tumour growth (Borem et al., 2003).

Viral vectors however are more promising than non-viral delivery methods. Table 3 showcases several viral vectors currently in use. After much development, advancement towards the creation of innovative viral vectors with immunogenicity delivery with greater efficiency and low genotoxicity is becoming a reality with adeno-associated virus (AAV) being at the forefront. AAV is believed to be a beneficial vector due to its distinct structures, allowing novel treatments of neurodegenerative diseases in the CNS (Qu et al, 2019).

Parkinsons disease GDNF AAV2 Putaminal 25 NCT01621581 Phase I, active not recruiting Convection-enhanced delivery method was performed to infuse virus vectors into the brain

Vaccinia virus 25Kb Non-integrating Dividing and non-dividing cells Transient (only last short time) Huntingtons Disease (HD) is an autosomal dominant disorder which is passed from an affected parent to half their offspring, causing progressive degeneration/death of striatal neurons. Several studies have been conducted with one study highlighting that a SIRT3 gene, once delivered by an AAV vector, could prevent neurodegeneration from occurring within HD mice (Dufour et al., 2014). The delivery of SIRT3 prevented mitochondrial oxidative stress from occurring and thus maintained neuronal bioenergy. By 2018, laboratory and preliminary clinical research suggests that clinically applied treatment in regard to HD is now plausible (Qu et al., 2019).

Canavan disease is an autosomal recessive neurodegenerative disease which is caused by diffused spongiform white matter degeneration in the brain alongside dysmyelination and intramyelinic oedema. The condition arises due to aspartoacylase being inactive, thereby allowing Nacetylaspartate to toxically accumulate (Qu et al, 2019). Studies show that gene therapy would be beneficial to the patient, particularly when younger as, during a clinical trial in 2012, 13 Canavan disease patients received AAV2-aspartoacylase intraparenchymally with the long-term effects being recorded. All 13 patients responded well to treatment with no serious side effects developing (Ahmed and Gao., 2013). Therapeutically, the amount of Nacetylaspartate in the brain had decreased, seizures became less frequent and progressive brain atrophy was delayed. As a result, its recommended that early therapeutic intervention is taken to prevent the disease from escalating (Qu et al., 2019). Despite AAV being highly successful, there have been other trials in which the end results portrayed AAV as ineffective but, some of the trials potentially lacked a complete thorough knowledge about the disease itself. Clinically, AAV has other opposition, particularly neutralising antibodies. Any pre-exiting antibodies of AAV have caused an increasingly small limit of AAV vector usage in clinical gene therapy. (Ortolano et al., 2012; Rapti et al., 2012).

With gene therapy still a recently novel scientific field, it has a promising future despite its unfortunately morbid start. On September 18, 1999, Jesse Gelsinger, whom was ornithine transcarboamylase deficient, an enzyme essential for ammonia metabolism, died after the gene therapy he received caused him to undergo hepatic and respiratory failure. After a temporary moratorium on human clinical trials, the FDA allowed further human-related investigations to continue upon the advancement of Biotechnology practises such as vector construction for cloning and improvements in the transformation of competent cells by protocols like PURELINK (Borem et al., 2003). Abina et al. (2015) had a major breakthrough in gene therapy by identifying the genetic anomalies of the rare, congenital immune and platelet deficiency, X-linked Wiskott-Aldrich syndrome (WAS), therapeutically correcting it by injecting a lentiviral vector with the target WASp gene into the cells before being reinjected into the patients whom, at the time, had been undergoing chemotherapy so that the newly corrected stem cells would differentiate instead of their defective cells. After nine months, the six subjects immune systems were restored, and their clinical condition improved. With the scientific community captivated by the wonders of gene therapy and its recent successes within the last decade, particularly with viral-delivery vectors, its only a matter of time before all of the technical aspects and scientific details are developed and gene therapy becomes a staple in genetic disorder treatment.

References

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