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.

Cell

Most cells in our bodies contain DNA inside the nucleus.

DNA

The DNA in the nucleus of a cell is referred to as its genome. The human genome contains about 20,000 genes.

CRISPR-Cas9 GENE THERAPY Leber Congenital Amaurosis

CRISPR-Cas9

Genetic Medicine

HHMI Investigator, Jennifer Doudna talks about the role of CRISPR-Cas9 technology in research and medicine.

CRISPR-Cas9 is a technology that allows scientists to change a cell's DNA at a precise location. 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.

Learn more

diagram of crispr-cas9 technology

CRISPR-Cas9 consists of the enzyme Cas9 and a guide RNA. By designing different guide RNAs, scientists can target the enzyme to any sequence.

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 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. As a result, on rare occasions 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. If the DNA break is repaired correctly, CRISPR-Cas9 will cleave the sequence again. Eventually, the repair process will introduce a mutation in the gene of interest.

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 many copies of 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 in other applications.

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 locus within the prokaryotic genome.

Gene Therapy Case Study: Leber Congenital Amaurosis

Genetic Medicine

Gene therapy uses modified viruses to deliver therapeutic genes to cells.

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.

Learn more

gene therapy diagram

The basic process of delivering gene therapy to a patient's cells.

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 adenoviruses. 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 their genomes into the host cell genome. Lentivirus vectors are commonly used integrating viral vectors. While integration reduces the need for follow-up treatments, the technique poses the risk of introducing mutations at the site of integration.

Case Study

diagram of a cross section of the human eye

Diagram of a cross section of the human eye. Light enters the eye through the pupil and hits the retina at the back.

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.

Learn more

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 Switches Case Study: Sickle Cell Disease

Genetic Medicine

diagram of a gene switch and activator

Gene switches regulate the expression of genes by binding to different proteins that affect the activity of RNA polymerase.

One class of genetic technologies targets noncoding, regulatory DNA sequences in the genome, referred to as gene switches. These switches bind regulatory proteins that turn the transcription of genes on or off. Depending on the regulatory proteins present in a cell, genes can be expressed in different tissues, at different stages of development, or in response to certain 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.

Case Study

illustration showing flow of healthy and sickle-cell blood cells

In patients, the protein subunits that make up hemoglobin stick together to form long, inflexible rods, giving blood cells a sickle-like shape.

Sickle cell disease is an autosomal recessive condition that can be caused by mutations in the gene for β-globin (HBB). HBB is 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.

Learn more

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 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. Scientists have not yet tested this treatment strategy in humans.

Exon Skipping Case Study: Duchenne Muscular Dystrophy

Genetic Medicine

illustration exon skipping

A mutation that blocks translation results in no protein, or a nonfunctional protein fragment. Exon skipping allows a shortened, but partially functional protein to be produced.

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.

Learn more

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.

Case Study

Duchenne Muscular Dystrophy is an X-linked, progressive disease that almost always affects males.

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.

Learn more

illustration of healthy muscle cells and muscle cells with Duchenne's muscular dystropy

The muscle cells of children with DMD become progressively damaged, and muscles, including the heart, eventually stop working.

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, additional trials and monitoring are needed to establish a clear clinical benefit of the drug.

RNA Interference Case Study: Huntington’s Disease

Genetic Medicine

illustration of RNA interference

Small interfering RNA (siRN) is incorporated into a protein called RISC. Once the RISC finds a complementary sequence in a messenger RNA (mRNA), it cleaves the mRNA.

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 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.

Learn more

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.

Case Study

illustration of cross-seciton of healthy brain and one with Huntington's disease

Huntington's disease affects the whole brain, but cells in the basal ganglia are particularly affected. As the basal ganglia shrink (atrophy), the ventricles take up more space.

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 currently underway to test the safety of an RNA interference therapy for HD. It is designed to reduce production of the mutant HTT protein by destroying the mutant HTT mRNA.

Learn more

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.

Small Molecule Drug Case Study: Cystic Fibrosis

Genetic Medicine

table comparing Aspirin, Gleevec and Ivacaftor

Small molecule drugs, like aspirin, Gleevec, and ivacator, make up over 90 percent of the drugs on the market today.

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.

Learn more

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.

Ivacator is a new small molecule drug that can treat certain mutations that cause cystic fibrosis.

Case Study

Patients with cystic fibrosis suffer from frequent lung infections.

Cystic fibrosis (CF) is an autosomal recessive disease caused by any one of more than 2,000 mutations in a 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.

Learn more

illustration of normal and mutated cell membranes

The CFTR channel allows the transport of chloride ions across the membrane of cells that normally produce a clear, watery fluid. Mutations that result in either no CFTR channel or a dysfunctional channel cause these negatively charged ions to accumulate inside cells, drawing water from the outside and leaving behind an abnormally thick mucus.

In patients with CF, the CFTR protein may be absent, dysfunctional, or functional but present in low numbers. The most common mutation, called Phe508del, is a deletion of one amino acid (phenylalanine) at position 508 in the CFTR protein. About half of all CF patients are homozygous for this mutation.

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 and lumacaftor, used in combination, are beneficial to CF patients with certain CFTR mutations. However, when used on their own, these drugs do not seem to benefit these patients.

About

This interactive uses the central dogma as a model for exploring how modern molecular biology technologies can be used to treat genetic diseases. The interactive can be explored by scrolling down the page to visualize the steps in the central dogma pathway. Clicking on the pink "+" signs opens up a window with information about a treatment strategy that targets that step, and a case study of a genetic disease for which that strategy has been shown to work either in experimental models or human clinical trials. The interactive can also be explored by clicking the menu topics across the top.

The case studies and technologies were chosen based on some of the patients featured in the hour-long documentary The Gene Doctors, an HHMI Tangled Bank Studios Production in association with RCSR Productions. The interactive includes several short video clips from the program.

Drug information is current as of August 2017. As this is a rapidly advancing area of science, we will update this interactive frequently.

Credits

Producers

Aileen O'Hearn, PhD, HHMI
Fabian de Kok-Mercado, MA-CMI, HHMI

Writing and Editing

Aileen O'Hearn, PhD, HHMI
Laura Bonetta, PhD, HHMI
Satoshi Amagai, PhD, HHMI
Ann Brokaw, Rocky River High School, Rocky River, OH

Scientific Reviewers

Chris Gunter, PhD, Emory University
Kevin Davies, PhD

Fact Checking

Christina Wyman, MA

Art Direction & Central Dogma Graphics

Fabian de Kok-Mercado, MA-CMI, HHMI

Additional Graphics

Heather McDonald

Web Editor

Chris Vargas, HHMI

Executive Producer

Dennis Liu, PhD, HHMI

Design and Development

kapow, inc.

Educator Resources

The central dogma of molecular biology is a fundamental concept in the study of biology, found in all major biology curricula (NGSS, non-NGSS state-level life science, AP, IB, and undergraduate Vision and Change) and biology textbooks. Educators can therefore use this interactive in a variety of courses, ranging from first-year high school biology to undergraduate molecular biology electives. The connection to genetic techniques and their applications extends its use to biotechnology-focused courses.

The interactive can be used to teach or review:

  • the steps involved in eukaryotic gene expression;
  • the connection between genetic mutations and phenotype;
  • the causes and symptoms of different monogenic diseases; and
  • methods and tools used in genetic research, such as CRISPR, RNAi, and gene replacement.

Exploration of this interactive ties to several science practices including the use of models and representations, engagement in scientific questioning, construction of evidence-based explanations, and relating knowledge across scales and concepts. The interactive will also emphasize key crosscutting concepts that help students better understand core ideas in science including the importance of structure and function; scale; and cause and effect.

Educators should keep in mind that the flow of information from DNA to RNA to protein is a dynamic process that involves many more steps and molecules than are shown in this interactive. In addition, there are exceptions to this orderly flow. For example, scientists have discovered that noncoding RNA and retroviruses are capable of reverse transcription, in which genetic information flows from RNA to DNA. Educators might also remind students that, except for cancer, diseases featured in this interactive are rare monogenic diseases, caused by mutations in single genes. It will be more challenging to apply these technologies to more common diseases caused by a combination of genes and environmental factors. In most cases, more than one treatment strategy or technology may be applied to any particular disease.

Curriculum Connections

Curriculum Standards
NGSS LS-1-1, LS-3-1, LS-3-2
AP Biology 1.B.1, 3.A.1, 3.A.2, 3.A.3, 3.C.1, 4.A.1
IB Biology 2.4, 2.5, 2.6, 2.7, 3.1, 3.2, 3.4, 3.5, 7.2, 7.3, B.4

Key Concepts

  • Identifying the mutation that causes a disease can provide a way to treat it.
  • In most cases, genetic information flows from DNA to mRNA to protein; genetic diseases can be treated at different steps in this pathway.
  • The three-dimensional structure of a protein affects its function.

References

CRISPR

You Lu (Sichuan University), "PD-1 Knockout Engineered T Cells for Metastatic Non-Small Cell Lung Cancer," accessed July 31, 2017, https://clinicaltrials.gov/ct2/show/NCT02793856?term=crispr&rank=6.

Leber Congenital Amaurosis

James W.B. Bainbridge et al., "Effect of Gene Therapy on Visual Function in Leber's Congenital Amaurosis," New England Journal of Medicine 358, no. 21 (May 22, 2008): 2231–39, doi:10.1056/NEJMoa0802268.

Oscar Francisco Chacon-Camacho and Juan Carlos Zenteno, "Review and Update on the Molecular Basis of Leber Congenital Amaurosis," World Journal of Clinical Cases 3, no. 2 (February 16, 2015): 112, doi:10.12998/wjcc.v3.i2.112.

Sickle Cell

CDC, "Data and Statistics | Sickle Cell Disease | NCBDDD | CDC," 2016, https://www.cdc.gov/ncbddd/sicklecell/data.html.

Christian Brendel et al., "Lineage-Specific BCL11A Knockdown Circumvents Toxicities and Reverses Sickle Phenotype," Journal of Clinical Investigation 126, no. 10 (September 6, 2016): 3868–78, doi:10.1172/JCI87885.

Huntington's Disease

Michael D. Rawlins et al., "The Prevalence of Huntington's Disease," Neuroepidemiology 46, no. 2 (2016): 144–53, doi:10.1159/000443738.

Holly B. Kordasiewicz et al., "Sustained Therapeutic Reversal of Huntington's Disease by Transient Repression of Huntingtin Synthesis," Neuron 74, no. 6 (June 2012): 1031–44, doi:10.1016/j.neuron.2012.05.009.

Ionis Pharmaceuticals, "Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of IONIS-HTTRx in Patients With Early Manifest Huntington's Disease - Full Text View - ClinicalTrials.gov," 2015, https://clinicaltrials.gov/ct2/show/NCT02519036.

Duchenne Muscular Dystrophy

Alan E.H. Emery, "Population Frequencies of Inherited Neuromuscular Diseases—A World Survey," Neuromuscular Disorders 1, no. 1 (1991): 19–29, doi:10.1016/0960-8966(91)90039-U.

Maria Kinali et al., "Local Restoration of Dystrophin Expression with the Morpholino Oligomer AVI-4658 in Duchenne Muscular Dystrophy: A Single-Blind, Placebo-Controlled, Dose-Escalation, Proof-of-Concept Study," Lancet Neurol. 8 (2009), doi:10.1016/S1474.

Cystic Fibrosis

Michael R. Kosorok, Wen-Hsiang Wei, and Philip M. Farrell, "The Incidence of Cystic Fibrosis," Statistics in Medicine 15, no. 5 (March 15, 1996): 449–62, doi:10.1002/(SICI)1097-0258(19960315)15:5<449::AID-SIM173>3.0.CO;2-X.

Kelly Kuk and Jennifer L. Taylor-Cousar, "Lumacaftor and Ivacaftor in the Management of Patients with Cystic Fibrosis: Current Evidence and Future Prospects," Therapeutic Advances in Respiratory Disease 9, no. 6 (December 2015): 313–26, doi:10.1177/1753465815601934.

Help

Browser and Device Support

This interactive is optimized for use on desktop and tablets and is optimized to work best with the Google Chrome web browser. Mobile and additional browser support are in development.

Navigation

Navigate through the Click and Learn by scrolling or using the Next and Previous navigation buttons. Explore types of genetic medicine and associated case studies by selecting the pink pulsating icons.

Feedback

For feedback and suggestions about the Click and Learn, email biointeractive@hhmi.org.