Scale: 5 nm
Genes are sequences of DNA that, for the most part, code for proteins. The flow of genetic information from DNA to RNA to protein has been referred to as the central dogma of molecular biology.
Mutations in genes can affect the resulting proteins and some mutations cause disease. To treat genetic diseases, scientists and doctors can intervene at different steps in the central dogma.
Scroll down to explore the steps in the central dogma pathway in a human cell. Click on the plus signs to explore a genetic medicine strategy that is being used to intervene at a particular step and a disease that could be treated using that strategy.
CRISPR-Cas9 is a technology that allows scientists to edit a cell's DNA, almost like a molecular scalpel. It is widely used in research and has great potential for therapy.
Treatments using CRISPR-Cas9 technology are being developed for several genetic diseases, including sickle cell disease and cystic fibrosis.
Scientists can use CRISPR-Cas9 to knock out a gene so that it is not expressed, or edit the gene to correct a disease-causing mutation.
CRISPR-Cas9 comprises a nuclease (an enzyme that cleaves nucleic acids) called Cas9, paired with an RNA fragment called guide RNA. The guide RNA is designed to bind to a specific DNA sequence in the genome of a cell. It guides Cas9 to that target sequence, where the enzyme cleaves the DNA, leaving a double-stranded, blunt-end break. Once the DNA is cut, researchers can take advantage of the cell's DNA repair machinery to add or delete pieces of DNA, or replace an existing segment with a DNA sequence they designed.
When the Cas9-guide RNA complex cleaves the DNA in a cell, the cell's machinery will recognize and repair the double-stranded DNA break using one of two repair mechanisms: nonhomologous end joining (NHEJ) and homology-directed repair (HDR).
NHEJ is the more frequently used, faster repair mechanism, because the cell does not use a template to join broken DNA ends together. If the DNA break is repaired correctly by NHEJ, CRISPR-Cas9 will just recognize and cleave the sequence again. Eventually, the repair process will introduce a mutation in the gene of interest. As a result, NHEJ can result in small 1- to 10-base pair insertions/deletions (indels). Such mutations can lead to a frameshift that knocks out that gene's function.
HDR is less error-prone and uses a homologous DNA template to repair the DNA break. Scientists can take advantage of this process by introducing an artificial template DNA in cells along with CRISPR-Cas9. The repair template includes a DNA sequence that scientists want to introduce at the site of the cleavage. After Cas9 cleaves the cell's DNA, the HDR mechanism repairs the break by copying the sequence from the template DNA. This mechanism can be used to repair a disease-causing mutation in a gene, change the function of a gene, or otherwise edit DNA.
The CRISPR-Cas9 technology is a simplified version of a natural defense mechanism of bacteria and archaea that protects them against invading viral pathogens. CRISPR stands for clustered regularly interspaced short palindromic repeats, and is named for a location within the prokaryotic genome.
Gene therapy is an experimental technology that allows researchers to provide functioning copies of genes to cells with disease-causing versions of those genes. The therapeutic genes can be delivered inside cells in a variety of ways. One common delivery method uses viral vectors—modified viruses in which harmful portions of the virus genome are replaced with the therapeutic gene. When a viral vector is introduced into the body, it infects target cells and releases its genome, including the therapeutic gene, inside. The cell's machinery then produces a functional protein from the introduced gene.
Viral vectors can be integrating or nonintegrating. Nonintegrating vectors deliver the therapeutic gene within a stand-alone genetic element within the cell, called an episome. Commonly used nonintegrating viral vectors include modified adeno-associated viruses, which do not normally cause disease in humans. Because the viral genome is not integrated into the host cell genome, the therapeutic gene is not transferred into all the daughter cells of newly dividing cells and the therapy must be periodically readministered.
Integrating vectors insert copies of their genomes into the host cell genome. Lentivirus vectors are commonly used integrating viral vectors: one advantage is that they can infect non-dividing cells, such as neurons. While integration reduces the need for follow-up treatments, the technique poses the risk of introducing mutations at the site of integration.
Leber congenital amaurosis (LCA) is a rare genetic disease, affecting 2-3 per 100,000 newborns in the US. It involves the cells of the retina and causes extreme far-sightedness or blindness at birth.
LCA can be caused by a mutation in any one of at least 20 known genes. The mutations disrupt processes by which cells of the retina convert light into nerve signals to the brain. Most cases are autosomal recessive. In 2008, scientists reported the first positive results of an experimental gene therapy in humans, targeting LCA patients with mutations in a gene called RPE65.
Gene therapy was successfully used to replace the mutated version of RPE65 in three patients with LCA in 2008. Scientists reported similar results with 20 more patients in 2017.
RPE65 produces an enzyme involved in processing a type of vitamin A, which the light-sensing photoreceptor cells of the retina—the rods and cones—require to function.
Scientists used an adenoviral vector to introduce the normal RPE65 sequence into the patients' retinas, improving their vision.
Gene expression is sometimes controlled by regulatory DNA sequences in the genome, referred to as gene "switches." These switch sequences are noncoding, and regulatory proteins can bind to them, turning the transcription of the gene on or off. Depending on the regulatory proteins present in a cell at any given time, genes can be expressed in different tissues, at different stages of development, or in response to specific stimuli.
Scientists have begun to explore ways of interfering with gene switches to alter gene expression. Such methods can be used either to prevent genes linked to diseases from being turned on, or to keep beneficial genes from being turned off.
Sickle cell disease (SCD) is an autosomal recessive condition that can be caused by mutations in the gene for β-globin (HBB). It encodes for HBB, one of the two subunits of adult hemoglobin, the protein that carries oxygen in red blood cells. People who inherit two copies of the mutation produce only abnormal HBB that result in sickle-shaped blood cells. The cells get stuck in small blood vessels, leaving body tissues starved for oxygen and causing pain, and, over time, organ damage.
Scientists are developing treatments that target the gene switch for fetal hemoglobin. Fetal hemoglobin production is normally turned off soon after birth; keeping it turned on would provide a source of functional hemoglobin in patients with SCD.
Fetal hemoglobin is expressed in the fetus. It has higher affinity for oxygen than adult hemoglobin, allowing it to extract oxygen from the mother's bloodstream. After birth, babies start producing adult hemoglobin and turn off fetal hemoglobin production at around 6 months of age.
Scientists have been able to maintain fetal hemoglobin production in adult mice. To do so, they took the stem cells that give rise to red blood cells and blocked production of BCL11A, a repressor protein for the gene switch for fetal hemoglobin. When the stem cells were reintroduced in mice, they gave rise to red blood cells lacking BCL11A. Without the repressor protein, these red blood cells continue to express fetal hemoglobin in adult mice. In 2019, a clinical trial modifying expression of the gene BCL11A in humans showed early success, and is now being tested on more patients with SCD.
Exon skipping is a technology that changes how the primary RNA transcript of a gene with a disease-causing mutation is spliced, removing the mutation from the resulting mRNA.
Scientists design a short segment of single-stranded RNA (called antisense RNA or antisense oligonucleotide) to bind to a sequence of the primary RNA transcript for a gene that is normally recognized by the cell's splicing machinery. When the antisense RNA is introduced in a patient's cells, it binds to the target sequence in the primary RNA transcript, causing the splicing machinery to "skip over" a segment of the transcript. Depending on how the antisense RNA is designed, one or more exons may be spliced out of the resulting mature mRNA, including the exon with the disease-causing mutation.
The RNA used in exon skipping is called an antisense RNA or antisense oligonucleotide because it is complementary to the RNA transcript, which is transcribed from a DNA strand referred to as the "sense" strand. Oligonucleotide means a string of nucleotides.
The antisense RNA is synthesized to bind to a specific sequence in an RNA transcript that is recognized by the large protein complex (the spliceosome) that carries out splicing. During splicing, the spliceosome removes introns from an RNA transcript and joins exons together. A bound antisense RNA will hide a spliceosome recognition sequence, thereby altering the splicing process and the resulting mature mRNA transcript.
Muscular dystrophy (MD) refers to a group of more than 30 diseases that cause progressive muscle weakness. The most common form of MD is a severe form called Duchenne (DMD), which is caused by mutations in a gene that codes for the protein dystrophin in muscle cells. Children with DMD produce little or no dystrophin, which normally functions to connect muscle fibers to surrounding tissues. They are typically wheelchair-bound by their teens and die in the second to fourth decade of life.
The exon-skipping drug eteplirsen can cause the exon containing the disease-causing mutation to be spliced out of the dystrophin mRNA, allowing cells to produce a shortened, yet partially functional, protein.
The gene that codes for dystrophin is one of the largest human genes, comprising 79 exons. Mutations in this gene cause both DMD and a milder form of the disease called Becker MD. The mutations that cause DMD—including deletions, duplications, and point mutations—cause the coding sequence to become "out-of-frame" and end protein translation prematurely. These mutations result in no protein or a nonfunctional protein being made.
The mutations that cause Becker MD, on the other hand, do not disrupt the reading frame. They result in shortened dystrophin proteins that are at least partially functional. Patients with Becker MD are able to walk into late adulthood and have a normal lifespan.
The Becker MD mutations suggested to scientists that if they could restore the proper reading frame of a mutant dystrophin gene in DMD patients, they could produce partially functional proteins, and ameliorate some of the disease symptoms.
Eteplirsen does exactly that. It consists of an antisense RNA that binds to splicing signals on exon 51 of the dystrophin gene and causes the exon to be spliced out of the mRNA. For DMD patients with mutations in this region, removing this exon restores the proper reading frame so that a partially functional protein is produced.
Eteplirsen was approved by the FDA through its accelerated approval pathway; however, new studies have since shown that eteplirsen can cause respiratory and cardiac problems in some patients with DMD.
RNA interference (RNAi) technologies involve small RNA segments that target various mRNAs for destruction, reducing the expression of certain genes.
The RNAi method that's most well-studied for its potential in therapy uses small interfering RNA (siRNA). Scientists synthesize a short double-stranded RNA segment with a sequence complementary to that of a target sequence and introduce it into the body. The double-stranded siRNA is taken up by cells, where it is recognized as being foreign, possibly from a virus, and cleaved into smaller pieces. Single RNA strands from the siRNA pieces are then incorporated into a cellular protein complex, called the RNA-induced silencing complex (RISC). The siRNA guides the RISC to an mRNA with a complementary sequence and cleaves it. By synthesizing different siRNAs, scientists can potentially silence any gene.
Two major types of RNA molecules can potentially be used as RNAi therapies: small interfering RNA (siRNA) and microRNA (miRNA). Cells normally produce siRNAs as a defense mechanism against the double-stranded RNA genomes of some viruses. Cells produce miRNAs as a mechanism for regulating gene expression.
Scientists can synthesize both siRNA and miRNA molecules artificially to shut down the expression of certain genes. In general, siRNAs are highly specific, each with only one mRNA target, whereas miRNAs have multiple targets. To elicit an RNAi response, the siRNA must be fully complementary to its target mRNA and the miRNA only partially complementary.
One of the biggest hurdles for RNAi therapies is to design the sequences of the siRNAs and miRNAs to avoid affecting the expression of unwanted targets (off-target effects) or stimulating immune responses.
Huntington's disease (HD) is an autosomal dominant disease caused by mutations in a gene called huntingtin (HTT), which is required for normal nerve cell function. Mutations in the HTT gene can cause an abnormal protein to be made that causes brain cells to die over time. Disease symptoms typically start in adulthood and worsen over time. They include dementia, gradually deteriorating motor function, and eventually death.
Clinical trials are testing an RNA interference therapy for HD designed to reduce production of the mutant HTT protein by destroying the mutant HTT mRNA. So far, the therapy has been able to lower levels of mutant HTT in the spinal fluid of some patients. Larger trials are needed to see if the treatment can improve progression of HD and its symptoms.
The mutation that causes HD involves a segment within the HTT gene that contains repeated sequences of three nucleotides: cytosine, adenine, and guanine (CAG). The triplet CAG codes for the amino acid glutamine.
Normally, the CAG repeat occurs around 10 to 35 times within the HTT gene, but the number of repeats is expanded in patients with HD. The expansion results in proteins with abnormally long stretches of glutamines, which cause proteins to "stick" together and accumulate in nerve cells, eventually interfering with normal cell operations.
Individuals with 36 to 39 CAG repeats in their HTT gene may or may not develop symptoms of disease, whereas people with 40 or more CAG repeats almost always do. The number of repeats is inversely associated with age of disease onset, meaning that the more repeats that are present, the earlier symptoms will likely start.
A therapy that can lower the expression of the mutated HTT RNA, and therefore reduce the level of mutated HTT protein, may help delay the age of disease onset or lessen the symptoms of HD.
Small-molecule therapies comprise an incredibly diverse group of chemical compounds of low molecular weight that are synthesized in the lab. Because of their small sizes, these molecules can easily be taken up by cells and may be administered to patients as pills or by injections. Small-molecule drugs may interact directly with disease-causing proteins, or through other molecules. Some small-molecule drugs block the negative effects of disease-causing proteins, whereas others restore their proper functioning.
With a molecular weight of about 180 g/mol, or 180 daltons, acetylsalicylic acid (ASA), the active ingredient in aspirin, can be taken as a pill that dissolves in the gastrointestinal tract and is then absorbed into the bloodstream. From there, it can reach almost any cell in the body. ASA binds to one of the enzymes involved in causing inflammation: cyclooxygenase-2.
Gleevec is a small molecule that binds to the active site of a mutated enzyme called BCR-ABL that is active in some cancer cells. Gleevec prevents BCR-ABL from binding its regular substrate, stopping cancer from growing.
Ivacaftor is a new small-molecule drug that can treat certain mutations that cause cystic fibrosis.
Cystic fibrosis (CF) is an autosomal recessive disease caused by any one of more than 2,000 mutations in the CFTR gene that codes for the cystic fibrosis transmembrane conductance regulator (CFTR). This protein functions as a channel that transports chloride ions across the membranes of cells that line airways, glands, and the digestive tract. Mutations prevent the channel from functioning properly and lead to cells producing a thick, sticky mucus that can obstruct airways and glands, providing a breeding ground for life-threatening infections.
The U.S. Food and Drug Administration (FDA) approved two small-molecule treatments for the most common CF mutation.
In patients with CF, the CFTR protein may be absent, dysfunctional, or functional but present in low numbers. The most common mutation, called F508del, is a deletion of one amino acid (phenylalanine) at position 508 in the CFTR protein. Approximately 41% of patients with CF have one copy of this mutation combined with a different mutation, while another 45% of patients have two copies of F508del.
The deletion causes defective CFTR protein folding and processing, resulting in minimal amounts of CFTR reaching the cell surface; for the few channels that do manage to get to the surface, the mutation also disrupts channel opening. Together, these effects lead to minimal chloride transport.
A small molecule called lumacaftor improves processing of the mutant CFTR protein, increasing the amount of protein in the cell membrane. Another small molecule called ivacaftor increases the amount of time that CFTR channels remain open.
Clinical studies have shown that ivacaftor on its own only benefits patients with a group of specific CFTR mutations, while lumacaftor does not appear to be an effective treatment on its own. However, ivacaftor and lumacaftor used in combination with each other are beneficial to patients with the F508del CFTR mutation.