2.2.1 Gene Therapy

In Lesson 2.1, you explored the science behind genetic testing and screening. You saw how DNA sequencing technology and bioinformatics have opened the door to identifying specific genes in our genome. Faulty genes are often the cause of many devastating illnesses and disorders. But what if scientists could go into our cells and replace, repair, or remove a gene that is malfunctioning? With the functional copy of the gene, a functioning protein could be produced and the disease could be eliminated. It may sound like science fiction, but scientists have already opened the door to molecular treatments, generally referred to as gene therapy, that can be used to correct a person’s genome. Doctors and medical researchers are experimenting with these tools to treat disease caused by gene mutations. To accomplish this goal, scientists have to get the right gene to the right cells in the right tissue. This is not an easy task. Many different molecular tools and delivery systems, known as vectors, are being used and investigated for gene therapy. Viruses are the main vectors currently being used in human gene therapy clinical trials. The idea of using viruses to deliver treatment may sound frightening, but these tiny pathogens can be an effective way to target our cells and insert new genes. Genome editing is another promising technique that uses tools in the cell to cut DNA and replace mutated genes. The process can remove viral genes that have been inserted in the human genome and cause infectious diseases. To prevent the need to alter each cell in the body, genome editing can be used to alter the DNA in stem cells that reside in the person's body that will produce healthy cells. Remember that stem cells are specialized cells that have the ability to develop into many different types of cells. While genome 1 editing has the potential to revolutionize gene therapy, it is several years away from being used in humans as a commonly prescribed treatment. CAREER CONNECTIONS Dr. Shoukhrat Mitalipov Embryologist Shoukhrat Mitalipov, director of the Oregon Health and Science University (OHSU) Center for Embryonic Cell and Gene Therapy, was recognized in 2018 for his work toward advancing the use of gene-editing tools to prevent inherited disease. Watch as Dr. Mitalipov discusses the work being done in his lab using breakthrough gene therapy techniques to cure disease and his advice for becoming a scientist. In this activity, you will investigate the different techniques used for gene therapy and how they work. You will explore current research on gene therapy, complete case studies, and gather evidence to showcase the benefits and potential risks of the different techniques. ● Computer with internet access and a diagramming tool 2 Procedure At its core, gene therapy seeks to modify one or more genes to alleviate the symptoms caused by a genetic disorder. However, the approach varies. In general, there are three strategies: ● INSERT: A functional gene is given to a patient, providing their body with the means to make a functional protein. The defective gene is still present but its effects are masked by the functional gene. ● DISABLE: A dysfunctional gene is disabled, eliminating the impact of the protein ● REPAIR: A dysfunctional gene is repaired so that it is able to produce a functional protein. List 2 genetic diseases that have FDA approved gene therapy. Research to identify the faulty gene(s) and complete the chart below. Disease Name of Treatment[s] Gene & Chromosome Locus [if known] LCA Luxturna RPE65, Chromosome 1 SMA Zolgensma SMN1, Chromosome 5 Explain why your selected genetic disorders would be good candidates for gene therapy? Both are good candidates because they are single-gene defects (caused by mutations in a single-gene), which means replacement will be a lot easier. 3 Explore the tools used to genetically engineer tissues. These tools are used to modify genes and in turn, the proteins they produce. Complete the table using the information presented in the Genetic Engineering slideshow. Vector Max. Gene Size and Nucleic Acid Type Cell Specificity Triggers Immune Response (Y/N) Host Cell Genome Integration (Y/N) Efficiency Plasmid Any Size DNA None N N Less Efficient Liposome Any Size DNA None N N Less Efficient Herpes Virus 20 kb DNA Nervous System Y N Less Efficient Adeno-associat ed Virus 5 kb DNA Variety of Cell Types N Y Efficient Adenovirus 7.5 kb DNA Variety of Cell Types Y N Efficient Retrovirus 8 kb RNA Dividing Cells Y Y Less Efficient Lentivirus 8.5 kb RNA Variety of Cell Types N Y Efficient As you have seen, there are a variety of vectors, but they fall into two general categories: viral and non-viral. No single vector is ideal for every genetic disorder; each has its benefits and limitations. As with all medical procedures, you need to customize the therapy to the patient and balance benefit with risk. 4 Additionally, the strategy for using gene therapy varies. For example, scientists may choose to introduce the vector directly into a patient. This is called in vivo therapy. Or they may choose to extract patient cells, introduce the vector to the cells in the lab, and then reintroduce the cells into the patient. This is in vitro therapy. What do you think are some pros and cons of in vivo and ex vivo therapy? List your answers in the space provided. in vivo ex vivo Pros Direct delivery into the patient’s body, less invasive. Faster and simpler, reducing overall treatment cost and time. The precision is very high, allowing the scientist to optimize the genetic modification before introducing the cells into the patient. Scientists have more control over the process. Cons Target precision is limited, and the immune system may attack the viral vector. Doses are often limited, mostly for systemic diseases. The therapy is invasive, requiring cell extraction. It is very time consuming, and much more expensive than in vivo therapy, due to cell harvesting and culturing. Gene Therapy Case Studies You will use what you have learned so far to complete two case studies. In the first study, beginning at step 6, you will follow the process to design a gene therapy for a patient with hemophilia. In the second case study, beginning at step 9, you will independently design a gene therapy for a new patient, Alec Faustus. Create a graphic organizer summarizing what you have learned about gene therapy. Include the diseases and the faulty gene(s) you identified in step 2 as an example. Also include the CRISPR-Cas9 genome editing system and summarize what you have learned about genome editing and how it can 5 be used to treat the disease that you chose. Use the information found in step 11 for CRISPR details. Place your concept map in the space provided. Answer Conclusion questions 1 and 2. Alec Faustus is an eight-month-old male experiencing some concerning symptoms, including swollen joints and excessive bleeding from small cuts and scrapes. Alec’s doctor recommended he have his DNA tested to screen for a genetic disorder. As Alec’s doctor, you have received his DNA testing results, which are shown below. 6 To explore the cause and effects of Alec’s genetic disorder, interact with this tour of models. Review the associated text in each chapter, and review the anatomical labels. Use the information found to complete the following table: Name of the mutated gene and protein. F8, Serine to Asparagine Location where this protein can be found. Symptoms of an individual with hemophilia experiences and why these occur. This protein can be found in the liver. Symptoms include blood being unable to clot, which is dangerous when the patient is bleeding, even a little. What is occurring in the cells? The cells send a protein into a bloodstream 7 to clot blood and stop bleeding. Design a gene therapy strategy for Alec. Place your information in the table below. How large is the gene the vector needs to carry? The gene is 2600 bp. Modification of which tissue would result in the production of a functional gene and alleviation of Alec’s symptoms? Tissue in the liver would result in the production of a functional gene and alleviation of Alec’s symptoms. Which vector will target the identified tissue and carry the gene of interest? Lentiviral will target the identified tissue and carry the gene of interest. Answer Conclusion questions 3 and 4. Since the late 1990s, scientists have been researching molecular systems that can accurately target specific DNA sequences to correct mutated DNA with more precision than traditional gene therapy. With traditional gene therapy, there is a chance that a new gene can be inserted in the wrong 8 location and the viral vectors can elicit an adverse immune response, which can lead to serious health concerns. The most current genome editing technique is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). While the name seems complicated, CRISPR refers to the short, palindromic repeated DNA sequences found in the genome of bacteria in which the system was discovered by researchers. CRISPR is coupled with the Cas9 restriction enzyme to edit genes and is known as the CRISPR-Cas9 system. CRISPR-Cas9 Goes Viral CRISPR-Cas9 is a powerful genomic engineering technology that enables scientists to change the DNA of animals, plants, and microorganisms with precision. In 2020, Emmanuelle Charpentier and Jennifer A. Doudna received the Nobel Prize in Chemistry for their discovery of the CRISPR/Cas9 genetic scissors that have revolutionized genome editing. Watch as Jennifer Doudna, Martin Jinek, and Emmanuelle Charpentier describe their collaboration and experiment that led to the use of CRISPR as a genome editing technology. CRISPR Video Watch the following 3 videos about the CRISPR-Cas9 genome editing system. As you watch the videos, answer the following questions and record the answers in the table below. How does CRISPR target the gene of interest? CRISPR targets the gene of interest by using RNA that recognizes the sequence. 9 What role does Cas9 play in the CRISPR system? Cas9 cuts the gene and inserts the new one in the CRISPR system. Which diseases are candidates for treatment for the CRISPR-Cas9 system? Diseases that are candidates for treatment for the CRISPR-Cas9 system are inherited diseases like CF. How can CRISPR-Cas9 be tailored to target different genes? CRISPR-Cas9 can be tailored to different genes by using different RNA that recognizes different sequences. Answer Conclusion questions 5 and 6. You will be placed in a small group. Half your group will read 4 of the following articles that describe evidence against the use of gene therapy. The remaining members of the group will choose and read four of the following articles that argue in favor of genetic engineering: 10 Read the articles and record evidence in the space below that supports the viewpoint you were asked to research. Present your evidence to the class for discussion. In my article, “Gene Therapy Experiments Improve Vision in Nearly Blind,” there is ample evidence to show that genetic engineering is good for people. In the article, patients with Leber Congenital Amaurosis had their vision restored, with two of them even being able to read a few lines of an eye chart. This provides evidence that severe vision loss can improve disorders. The study also reported no side effects from the therapy, which shows that when carefully controlled, it can be safe for patients. The research shows promising results, and more doses in younger patients could lead to outcomes even better. The National Eye Institute believes that the therapy could be even more refined and expanded to treat more disorders. Experts in the field, like Dr. Ronald Crystal, have expressed that these treatments give a “big shot in the arm” to gene therapy as a whole. The therapy gave significant improvement to the quality of life in patients like Steven Horvath, who can now navigate extremely better in low-light situations. Overall, these findings support the potential and future of genetic engineering, and the effects of reversing genetic mutations and diseases show why gene therapy as a whole is a positive. Answer the remaining Conclusion questions. 11 1. How do doctors and researchers decide whether a disease is a good candidate for gene therapy? Doctors and researchers decide whether a disease is a good candidate for gene therapy if the disease is a single-gene mutation and it affects a large population. 2. What are the advantages and disadvantages of using viral vectors for gene therapy? An advantage of using viral vectors for gene therapy is efficiency. A disadvantage of using viral vectors for gene therapy is an immune response that may be severe. 3. What factors must be considered when choosing a vector for a gene therapy trial? The factors that must be considered when choosing a vector for a gene therapy trial are if the vector can carry the gene, enter the body safely, and if it will cause a severe immune response. 4. A young boy suffers from Neurofibromatosis Type 1 (NF1), a genetic nervous system disorder that causes tumors to grow around nerve cells. The gene for NF1, approximately 8,400 base pairs long, is located on chromosome 11. What is the best candidate for a vector for gene therapy in this case? Provide evidence from your research to support your choice. The best candidate for a vector for gene therapy in this case is the Herpes Simplex Virus, which can hold a strand 20,000 base pairs long. It specifically targets nerve cells, which is what NF1 disrupts. 12 5. How does traditional gene therapy compare to the CRISPR-Cas9 genome editing system? Traditional gene therapy compares worse to the CRISPR-Cas9 genome editing system. CRISPR-Cas9 is more effective and efficient, while also not eliciting an immune response. 6. Genome editing has many advantages for treating and preventing diseases, however there are many ethical concerns. Explain one ethical concern related to genome editing. One ethical concern related to genome editing is the fact that people can change their babies to have advantages over other babies, and be able to control how their baby turns out. 7. Listen to the following podcast. Many athletes and administrators fear the widespread use of gene doping—the use of gene therapy to modify genes that improve athletic performance. Describe at least two genetic modifications that would improve the performance of an athlete. Make sure to mention the body system(s) affected as well as the specific goal of the therapy. Two genetic modifications that would improve the performance of an athlete are therapies to produce more hormones in the body, and a gene that can make drugs undetectable. These modifications target the skeletal system and the cardiovascular system for strength and oxygen delivery. 8. Explain how gene therapy can impact the future of humans as it relates to curing diseases and gene selection. Gene therapy can impact the future of humans as it relates to curing diseases and gene selection because people could start choosing and picking what genes to insert, which could make super humans. 13 9. With the discovery of CRISPR, we gained the ability to change the evolution of the human species. Explain how gene therapy can impact the future of humans as it relates to curing diseases and gene selection. Who decides the limits to use of the technology? Gene therapy can impact the future of humans as it relates to curing diseases and gene selection by making humans live a longer life, which would overpopulate the Earth and make billions of stronger humans. The people who decide the limits to the use of the technology should be the people and doctors to administer the technology, since they would know the effects. 14