4.5: Gene Therapy
Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. In theory, the introduction of a good gene will allow the organism to produce a functioning protein instead of the mutated or absent version caused by a disease. Gene therapy attempts have often involved introducing the good gene into diseased cells as part of a vector transmitted by a virus. The virus vector (containing the good gene) can then infect the host cell and deliver the foreign DNA to the diseased cells (Figure 2).
Figure 2: Gene therapy using an adenovirus vector can be used to cure certain genetic diseases in which a person has a defective gene. (credit: OpenStax Microbiology 4.4. CC-BY.)
CRISPR/Cas9
More recently, there has been much interest in correcting mutated genes using CRISPR/Cas9 technology that was discovered in bacteria. CRISPR stands for Clustered Regularly InterSpaced Palindromic Repeats sequences that exist in bacterial chromosomes. Bacteria insert foreign DNA into the CRISPR sequence and use that as a template or guide to survey the cell for that foreign DNA sequence and then cleave the foreign target DNA using the Cas9 enzyme. Thus, the CRISPR/Cas9 system is a form of bacterial immune system to recognize and cleave invading DNA from bacterial viruses.
CRISPR/Cas9 involves two essential components: a guide RNA to match a desired target gene, and Cas9 (CRISPR-associated protein 9), an endonuclease which causes a double-stranded DNA break, allowing modifications to the genome (see figure 3). Cas9 is the enzyme that cuts the target DNA. Scientists have discovered that CRISPR technology makes it possible to correct errors in the genome and turn on or off genes in cells and organisms quickly, cheaply and with relative ease. One of the most exciting applications of CRISPR/Cas9 is its potential use to treat genetic disorders caused by single gene mutations. Examples of such diseases include cystic fibrosis, Duchenne's muscular dystrophy (DMD) and haemoglobinopathies. Scientists have used CRISPR/Cas9 technology to restore cystic fibrosis transmembrane conductor receptor function in stem cells from cystic fibrosis patients. Researchers have also used CRISPR/Cas9 technology to restore muscle function by recovering dystrophin protein expression in a mouse model of Duchenne’s muscular dystrophy. The approach so far has currently only been validated in preclinical models, but there is hope it can soon be translated to clinical practice. Other potential clinical applications include gene therapy, treating infectious diseases such as HIV and engineering autologous patient material to treat cancer and other diseases. There has been increasing interest in the possibility of using CRISPR/Cas9 to modify patient-derived T-cells and stem/progenitor cells which can then be reintroduced into patients to treat disease, such as cancers.
Overview of CRISPR/Cas9
Figure 3
The CRISPR/Cas9 system. Clustered regularly interspaced palindromic repeats (CRISPR) refers to sequences in the bacterial genome. They afford protection against invading viruses, when combined with a series of CRISPR-associated (Cas) proteins. Cas9, one of the associated proteins, is an endonuclease that cuts both strands of DNA. Cas9 is directed to its target by a section of RNA. This can be synthesized as a single strand called a synthetic single guide RNA (sgRNA); the section of RNA which binds to the genomic DNA is 18–20 nucleotides. In order to cut, a specific sequence of DNA of between 2 and 5 nucleotides (the exact sequence depends upon the bacteria which produces the Cas9) must lie at the 3’ end of the guide RNA: this is called the protospacer adjacent motif (PAM). Repair after the DNA cut may occur via two pathways: non-homologous end joining, typically leading to a random insertion/deletion of DNA, or homology directed repair where a homologous piece of DNA is used as a repair template. It is the latter which allows precise genome editing: the homologous section of DNA with the required sequence change may be delivered with the Cas9 nuclease and sgRNA, theoretically allowing changes as precise as a single base-pair.
The therapeutic applications of CRISPR/Cas9 investigated have predominantly been directed at somatic cells. A particularly controversial issue surrounding CRISPR/Cas9 is that of gene editing in embryos. It has already been shown that CRISPR/Cas9 technology can alter the genome of human embryos3 which theoretically could prove useful in the preimplantation treatment of genetic diseases. However, any genetic modification of the germline would be permanent and the long-term consequences are unclear. Many oppose germline modification under any circumstances, reasoning that an eventual consequence could be non-therapeutic genetic enhancement.13It is clear that the ethical boundaries, within which CRISPR/Cas9 can be used, remain to be fully determined.
Microarrays
Some diseases, such as cancer, result from cells producing too much or too little of a protein. Microarrays are used to assess the levels of mRNA present in cells, which is indicative of the levels of proteins within cells. Microarray analysis is useful for the comparison of gene-expression patterns between different cell types—for example, cells infected with a virus versus uninfected cells, or cancerous cells versus healthy cells. Microarrays allow scientists to detect the expression of thousands of genes in one assay.
Microarray microscope slides, often called gene chips, are spotted with thousands of tiny spots containing different known DNA sequences. These DNA sequences act as probes. mRNA is isolated from cells (for example: mRNA could be isolated from a diseased cell and compared to mRNA from a healthy cell). The isolated mRNA is converted to a single stranded complementary DNA copy, cDNA, by using an enzyme called reverse transcriptase. The cDNA is labeled with a fluorescent dye and then allowed to bind to the microarray slide. cDNA will bind, or hybridize, to complementary DNA sequences on the microarray slide. Following time for hybridization to occur, the microarray slide is scanned for levels of fluorescence for each gene on the slide. Greater fluorescence associated with a gene indicates that the sample contained high levels of mRNA. No fluorescence for a specific gene indicates that the sample did not contain mRNA for that gene.
Often when performing microarrays, the cDNA from two different samples are labeled with two different fluorescent dyes (typically red and green). The fluorescently labeled cDNA samples are combined in equal amounts, added to the microarray chip, and allowed to hybridize to complementary spots on the microarray. Hybridization of sample genomic DNA molecules can be monitored by measuring the intensity of fluorescence at particular spots on the microarray. Differences in the amount of hybridization between the samples can be readily observed. If only one sample’s nucleic acids hybridize to a particular spot on the microarray, then that spot will appear either green or red. However, if both samples’ nucleic acids hybridize, then the spot will appear yellow due to the combination of the red and green dyes (Figure 4). When used in this way, microarrays can generate gene expression profiles that show differences in mRNA levels of many genes when comparing control/untreated cells to diseased or treated cells.
Figure 4: Microarray to compare mRNA levels. (a) The steps in microarray analysis are illustrated. Here, gene expression patterns are compared between cancerous cells and healthy cells. (b) Microarray information can be expressed as a heat map. Genes are shown on the left side; different samples are shown across the bottom. Genes expressed only in cancer cells are shown in varying shades of red; genes expressed only in normal cells are shown in varying shades of green. Genes that are expressed in both cancerous and normal cells are shown in yellow. Microarray CNX OpenStax, CC BY 4.0, via Wikimedia Commons.
Questions for Review
- When a gene chip is used in a microarray, what is spotted onto the microscope slide to create the gene chip?
- If someone is trying to determine if a cell expresses a protein that is known to be expressed in cancerous cells, what molecule would be isolated from the cells to be used in a microarray?
- Reverse transcriptase is mixed with the isolated samples to generate what molecule?
- What allows us to visualize if the samples bind to the microarray?
- What color shows on the microarray if both samples have bound to a sequence on the array?
mRNA Vaccines
In the 1970’s there was much interest in introducing mRNA into cells as a method of gene therapy. In theory, the mRNA would be translated directly within the cells to produce a correctly folded and functional gene, thus treating the disease. However, mRNA produced in vitro was found to elicit a strong inflammatory immune response and did not produce good or long lasting gene expression.
mRNA molecules are infamously known to be degraded quickly in the lab and in the body. Scientists who studied mRNA molecules for use in gene therapy had difficulty delivering the mRNA to the target cells before it would get quickly degraded by the body. In the late 1980’s scientists were beginning to uncover that enveloping mRNA in lipids (fats), creating a fatty nanoparticle, allows the mRNA to be successfully delivered to target cells without being quickly degraded before the mRNA can be translated to protein.
In 1998, Dr. Katalin Karikó, who worked extensively on mRNA, and Dr. Drew Weissman, an immunologist, began to collaborate to investigate the potential use of mRNA to generate vaccines. In theory, they hoped mRNA could be safely delivered to cells, then the mRNA is translated in the cytoplasm of the cells to generate a specific protein (generally a protein that is part of or produced by a specific pathogenic bacteria or virus). The body then mounts an immune response against the bacterial/viral protein and that memory immune response protects the person against that specific pathogen if it is encountered in the future. Initially, when mRNA vaccines were injected into mice they suffered severe inflammatory responses and some of the mice died. But, in 2005, Drs. Karikó and Weissman, published results showing that altering one of the mRNA building blocks, nucleosides, allowed mRNA to be less inflammatory and safely used therapeutically. It was becoming clear to their scientific team that it was possible to practically and safely use mRNA therapeutically. Early studies investigated the use of mRNA vaccines to prevent Ebola virus, but there was limited funding and demand for mRNA Ebola vaccines.
The COVID-19 pandemic brought a tremendous influx of funding and motivation to further investigate the use of mRNA vaccines to prevent or treat illness. Several different teams worked to develop mRNA COVID-19 vaccines. Extensive clinical trials showed that COVID-19 mRNA vaccines are both safe and effective. As of 2022, two independently developed mRNA COVID-19 vaccines have received full FDA approval, these are the first mRNA products to receive full FDA approval. mRNA has proven to be a powerful life-saving tool in the generation of vaccines. mRNA can be relatively easily and quickly manipulated, which makes mRNA vaccines the ideal method for the generation of vaccines against new and emerging deadly infectious diseases.
- Sometimes children who receive the chickenpox vaccine will develop minor chickenpox blisters/rash at the sight of the injection because this vaccine contains a weakened form of the chickenpox virus (Varicella zoster virus). Is it possible for someone to contract COVID-19 infection as a result of receiving a COVID-19 mRNA vaccine? Explain why or why not.
- Some people are concerned about mRNA vaccines modifying human DNA within the nucleus. Does this happen? Explain why or why not.
- When the COVID-19 mRNA vaccine enters our cells it is translated to make the viral spike protein. Does this mean that our cells will make the spike protein for the rest of our lives?
- How are mRNA vaccines considered to be safer than some other types of vaccines?
Questions for Review
- Detergent is added to DNA extraction buffer to ____________.
- Why does DNA precipitate when alcohol is added to the DNA extraction buffer?
- In genetic engineering, scientists make a specific change in the ___________ of an organism.
- The CRISPR/Cas9 system originated from what organisms? What purpose does this system serve in these organisms?
- Microarrays detect differences in levels of __________ in organisms.
Testing Understanding Post-Lab
- Sickle cell anemia is a genetic disease caused by a mutation in a single gene. This mutation causes the hemoglobin protein to be misfolded and therefore not able to carry as much oxygen throughout the body. Explain how CRISPR/Cas9 could possibly be used therapeutically to treat this genetic disease?