10 Medical Biotechnology I Gene Therapy A Genetic

10 Medical Biotechnology I. Gene Therapy A. Genetic Disorders B. Gene Target Selection C. Gene Delivery Methods D. Viral vectors 1. Retrovirus 2. Adenovirus 3. Adeno-Associated Virus E. Nonviral Delivery Methods F. Gene Therapy Examples 1. First Gene Therapy 2. Lung Disease—Cystic Fibrosis 3. Liver Disease II. Clinical Trials III. Issues in Gene Therapy - Cause for Concern? Is Gene Therapy Safe? IV. Recent Gene Therapy Success V. New Approaches to Gene Therapy A. Spliceosome Mediated RNA Trans-splicing B. Triplex-Helix-Forming Oligonucleotide Therapy C. Antisense Therapy D. Ribozyme Therapy VI. Virotherapy VII. Stem Cells A. Therapeutic Cloning and Embryonic Stem Cells VIII. Vaccines IX. Tissue Engineering X. Xenotransplantation XI. Drug Delivery A. Biosensors B. Biotech Revolution: Nanotechnology

I. Gene Therapy A. Introduction to Gene Therapy. 1. Treating genetic diseases by transferring normal genes into cells to correct a disorder is more desirable than treating the disease with a drug that might not be a cure. 2. May impact acquired and genetic diseases, ranging from cystic fibrosis to AIDS. 3. Gene therapy trials continue to be approved, even though there have been some devastating results during some trials. 4. Two types of gene therapy: a) Somatic—using only the body’s cells to correct a disorder. b) Germ line—permanently modifying a gene in the reproductive cells. There is currently a moratorium (ban) on this gene therapy because of the possible ethical and safety implications.

B. Genetic Disorders. 1. Medical problems caused by a mutation in one or more genes on the chromosomes. The person can be born with the mutation or develop it. 2. Can be grouped into four categories: a) Single-gene changes—a mutation in one gene can result in a change in the protein product or possibly the elimination of the protein entirely. Sickle cell anemia is an example. b) Multigene disorders—also called “multifactor disorders, ” result from mutations in more than one gene, and sometimes along with environmental influences. Examples of these include heart disease, diabetes, and cancer. c) Mitochondrial disorders—affecting many organ systems, these diseases are caused by mutations in mitochondrial DNA. d) Chromosome abnormalities—complete chromosomes or large regions of a chromosome are missing, duplicated, or modified in some way. Down syndrome is an example.

C. Gene Target Selection. 1. Several factors must be considered for a disease to be a candidate for gene therapy: a) Only a single gene causing a genetic disorder can be a candidate (Table 10. 1). b) The normal and mutated genes must be identified and well-studied. c) The disease caused by the mutation must be well-understood. d) An approved protocol involving a gene delivery method must be available. e) The potential toxic effects of the gene or gene delivery vehicle must be examined, as well as whetherapy produces an immune response. f) The gene must be delivered to the correct cells. g) If therapy method calls for it, the gene must be integrated into the host chromosome so the gene is not destroyed in host cells. h) The gene must be turned on (transcription) at the right time, and is also regulated properly.

D. Gene Delivery Methods. 1. There are two major strategies for the insertion of genes into cells: a) Ex vivo gene therapy: (1) Cells are removed from the body. (2) The gene of interest is inserted into them. (3) The cells are cultured for reproduction. (4) The cells are returned to the body. (5) An advantage over in vivo gene therapy is that rejection does not occur if the person’s own cells are used. (6) The transplantation of the cells is the biggest technical problem. b) In vivo gene therapy: (1) The gene is inserted directly into cells within the body. (2) Vectors such as viruses are used to target the DNA to specific cells. (3) Technical difficulties: (a) The transferred gene is unstable and the product produced temporarily. (b) The methods are not as controlled as ex vivo gene therapy because cells are not removed from the body.

E. Viral Vectors (Figure 10. 1) 1. Retrovirus. a) b) c) d) 2. Adenovirus. a) b) c) d) e) 3. RNA viruses that only infect dividing human cells. DNA of interest can only be up to 8 kb in size. Site of integration into host chromosomes occurs randomly. Viral insertion could inactivate genes or elicit an immune response. Can infect dividing and nondividing cells. Can engineer proteins on the virus surface to target specific cells. DNA of interest can be up to 7. 4 kb and the protein will be highly expressed. Does not integrate into the genome, so there is low risk of mutation. Possible drawbacks: (1) Temporary protein production because no integration occurs. (2) The virus may replicate in host cells, killing them. (3) Gene products may cause the cell to divide abnormally. (4) The virus of gene products may cause an immune response. Adeno-Associated Virus. a) Infect dividing and nondividing cells. b) Need a helper virus to infect host cells. c) DNA of interest can be up to 5 kb. d) 95% of the time DNA integrates into a region in chromosome 19, reducing the chance of genes being activated or inactivated by the insertion of DNA.

F. Nonviral Delivery Methods. 1. Other methods include electroporation, microinjection, biolistics (the “gene gun”), and liposomes (membrane-bound spheres that contain the DNA of interest).

G. Gene Therapy Examples. 1. First Gene Therapy. a) Occurred at the NIH for treatment of severe combined immune deficiency (SCID), which is caused by a defective adenosine deaminase gene that causes the immune system to not function correctly. b) In 1990, a 4 -year-old girl received T-lymphocytes with the correct ADA gene. c) The therapy had to be repeated because the enzyme would be produced for only a few months and then stopped. d) Three years later more than 50% of the T cells in the girl contained the corrected gene, and in 2003 her body still actively produces ADA. e) A second trial occurred on an 11 -year-old girl, but her immune system developed a reaction against the virus, and only 0. 1% to 1. 0% of her cells produced ADA.

2. Lung Disease—Cystic Fibrosis. a) Caused by a defective ion transport molecule called the “CF transmembrane conductance regulator” (CFTR) in the plasma membrane of a cell, and affects airways, the intestines, and the pancreas (Figure 10. 2). b) People with CF have symptoms such as increased mucus production, bacterial infections in the lungs, and altered epithelial cell transport. c) For therapy to work, only a small amount of molecules need to be produced per cell. It has been successful in cell culture and in CF mice by liposomes. d) Spraying adenoviruses with the CFTR gene into the nose has had only temporary treatment, and does not treat distant organs such as the pancreas. e) Another alternative is spraying a DNA-liposome aerosol into the nose, but it may not affect distant organs.

3. Liver Disease. a) Transfected hepatocytes (liver cells) have not been inserted into the liver with much success, with only about 10% of the cells incorporating into the liver. b) In 1992, a 29 -year-old patient was treated for familial hypercholesterolemia (FH): (1) Causes cholesterol to build up in arteries, leading to heart disease. (2) Caused by a defective low-density lipoprotein receptor (LDLR) gene. (3) 250 grams of the patient’s liver was removed and cultured. (4) A retrovirus was used to infect about 25% of the cells. (5) The cells were returned to the liver through a catheter. (6) Tests confirmed that the cells were expressing the LDLR gene, and medication contributed to lowering cholesterol levels.

II. Clinical Trials A. After therapy has been tested on animals (called “preclinical trials”), it is then tried on humans. B. There are several types of clinical trials: 1. Diagnostic trial—identifies better tests for diagnosing diseases. 2. Treatment trial—tests new therapies or drugs. 3. Prevention trial—looks for new ways to prevent disease in people who have never had the disease. 4. Quality of life trial—looks to improve the well-being and quality of life of chronically ill patients. 5. Screening trial—finds ways to detect specific diseases or health conditions.

C. There are several phases to clinical trials (Figure 10. 3): 1. Phase I Trials—testing on a small number (twenty to thirty) of human volunteers to determine dosage limits, route of delivery, and to assess the procedure’s toxicity and safety. 2. Phase II Trials—determine effectiveness and collect additional toxicity and safety information with a larger group of people (100– 300). If therapy is effective and safe, the researchers begin phase III trials. 3. Phase III Trials—a much larger group of people is tested (1000– 5000). Information obtained from phase I and II clinical trials is incorporated, and a comprehensive investigation of therapeutic role of the drug is conducted. 4. Seek FDA approval—once Phase III trials are completed, an application to the FDA is made for approval. 5. Phase IV Trials—when therapy is approved, if any questions remain regarding safety and efficacy, as well as the use of the treatment, they are addressed in this final check before the drug is allowed to be released.

III. Issues in Gene Therapy A. Even though gene therapy has great potential, many issues still need to be resolved to determine the safety and effectiveness of therapy: 1. 2. 3. B. Who will be eligible to receive gene therapy? Should gene therapy be used to prevent disease in addition to treating disease? Who will have access to gene therapy? How will it be made available and affordable to those in need? Cause for Concern? Is Gene Therapy Safe? 1. Gene therapy is a new field that has many risks, as seen in these two cases below. 2. Jesse Gelsinger case: a) An 18 -year-old with ornithine transcarbamylase (OTC) deficiency. b) Participated in a gene therapy study even though he was otherwise healthy. c) The therapy involved adenovirus vectors. d) The vectors led to his death due to clotting disorders and organ failure. 3. SCID studies and Leukemia: a) Nearly thirty gene therapy trials were cancelled after two children treated for ADA deficiency developed leukemia. b) The retrovirus used inserted the DNA next to an oncogene (a gene that can lead to cancer) in a white blood cell and cause them to grow uncontrollably.

IV. Recent Gene Therapy Success A. No one has completely been cured by gene therapy, but successes have many believing that gene therapy could be used for diseases that are difficult to treat. B. The first commercially licensed gene therapy: 1. In October 2003 in China, under the name Gendicine, after 5 years of clinical trials. 2. Treats head and neck squamous cell carcinoma. 3. An adenovirus vector containing the p 53 tumor suppressor gene; can be used along with chemotherapy and radiation therapy to increase effectiveness. 4. Only reported side effect is a low-grade fever.

V. New Approaches to Gene Therapy A. Why new approaches are needed: 1. Sometimes correcting the defective gene is not effective. 2. For example, a mutated gene can prevent a normal protein from functioning properly. This is called a “dominant negative gene” (Figure 10. 4) and cannot be corrected by simply inserting the correct gene. B. Spliceosome Mediated RNA Trans-splicing (SMa. RT) (Figure 10. 5). 1. Instead of correcting the gene, the region of m. RNA that is affected is repaired: a) An RNA strand that pairs with the intron (by base pairing) next to the mutated region (exon) of the m. RNA is introduced into cells. The RNA is genetically modified to contain the correct exon and a small region that binds to the neighboring intron. b) When the RNA strand binds to the intron, the duplex (section of double-stranded RNA) causes the spliceosome to cut and remove the intron and the defective exon from the m. RNA. c) The exons are joined, with the corrected exon ligated into the m. RNA, thereby generating a functional, mature m. RNA and protein.

C. Triplex-Helix-Forming Oligonucleotide Therapy (Figure 10. 6) 1. Synthetic triplex-forming nucleotides bind to target DNA regions, where they block transcription into m. RNA. 2. The single-stranded string of nucleotides, about fifteen to twenty-one bases in length, binds to the groove between the double strands of DNA where the mutated gene is used. 3. The triple helix that forms blocks transcription from occurring, so m. RNA will not be formed. 4. A correct gene can be introduced into cells to produce the functional protein.

D. Antisense Therapy (Figure 10. 7) 1. Targets the m. RNA of a mutated gene so that it cannot be translated into protein. 2. Is complimentary to the m. RNA that is used to code for the protein, called the “sense” m. RNA. 3. The steps of the method are: a) New, “antisense” RNA is introduced into cells that is complimentary to the m. RNA that is used to code for the protein, called the “sense” m. RNA. b) The antisense RNA binds to the sense m. RNA strands synthesized by the cell during transcription. c) The duplex RNA is blocked from translation into a protein, eliminating or dramatically reducing the production of a mutated protein. 4. May be used to treat diseases where there is a loss of control over gene regulation or if a gene is overexpressed. 5. The drug Genasense, which is in Phase II trials, targets the m. RNA for a protein called bcl-2, which is involved in the resistance of cancer cells to chemotherapy.

E. Ribozyme Therapy (Figure 10. 8). 1. m. RNA’s that act as enzymes that can cut m. RNA made by the cell. 2. They exist naturally in cells and have roles in m. RNA splicing (it is a part of the spliceosome) and the extension of the polypeptide during translation. 3. The steps of this method are: a) RNA is engineered to function as a ribozyme and to bind to the target m. RNA. b) The ribozyme is introduced into cells. c) The ribozyme binds to the target m. RNA encoded by the mutated gene. d) The target m. RNA is cut, keeping it from being translated into protein.

VI. Virotherapy A. Viruses can recognize and bind to a specific receptor on the host cell surface and usually kills the host cell. Each virus can attach to a different receptor, so viruses have cell specificity. B. Viruses are engineered to infect and kill tumor cells, leaving normal cells intact. C. Three different methods exist for virotherapy (Examples in Table 10. 2): 1. Transductional targeting to infect, reproduce, and kill tumor cells. Viruses from the killed cells will then infect other cancer cells. 2. Transcriptional targeting that uses engineered viruses that have a tumorspecific promoter linked to an essential virus gene. Although the virus can infect both normal and cancer cells, the gene turns on only in cancer cells and not normal cells. The virus kills the cancer cells. 3. Engineered viruses that render tumor cells more susceptible to chemotherapy.

VII. Stem Cells A. About Stem Cells: 1. Genetically engineered undifferentiated stem cells may have potential as gene therapy agents. 2. If stem cells containing a correct gene are inserted into a patient, the cells could divide and serve as a source of healthy cells to treat many diseases. 3. Scientists can isolate stem cells from a 5 -day-old mass of cells, called a “blastocyst, ” that develops into an embryo (Figure 10. 9). The cells are called “embryonic stem cells” and have the potential to develop into different cells of the body. 4. Goal of using stem cells is to treat damaged tissue by transplanting stem cells into a region of the body where they divide and differentiate into healthy tissue.

5. There are several types of stem cells: a) Embryonic—these cells are considered to be the most valuable type of stem cell because they can become almost any type of cell in the body. Can be obtained from in vitro fertilization (IVF) procedures. b) Fetal—found in fetal brain tissue and are a natural source of dopamine neurons. Must be harvested from prematurely terminated human fetuses or late-stage embryos. A human embryo is considered a fetus eight weeks after the egg is fertilized. Can possibly be used to treat Parkinson’s disease. c) Umbilical cord blood—multipotent stem cells. Even though they naturally become blood cells and immune system cells, they have the potential to become many different types of cells. Cause less rejection problems because they have not yet developed antigens that can be recognized by the immune system. d) Adult—multipotent adult stem cells that develop into cells of a specific type of tissue. Believed to be the least flexible in being able to develop into any type of tissue. Research today is focused on trying to get these cells to become different cell types.

6. Federal funding in the United States has been limited to nonembryonic stem cells since August 9, 2001. 7. Most experiments involve cells that are in culture and are harvested from bone marrow, peripheral blood, and umbilical cord blood. 8. Using bone marrow stem cells can treat diseases such as leukemia because they develop into white blood cells, by killing all of the abnormal bone marrow and white blood cells and replacing them with donor stem cells, which replace the damaged marrow and cells.

B. Therapeutic Cloning and Embryonic Stem Cells (Figure 10. 11). 1. Can obtain stem cells from embryos cloned in a similar fashion to Dolly, the cloned sheep. 2. Performed in the following manner: a) Nucleus is removed from an egg cell, and donor cells are extracted from the patient. b) Donor cells are cultured so that differentiation is reversed. c) Electrical pulse fuses the donor cell with the egg cell. d) The cell is induced to divide and form a blastocyst to extract stem cells from. 3. Stem cells generated this way are not rejected because they are an exact genetic match to the donor. 4. The research has brought up several questions: a) Should we be using human embryonic stem cells in research? b) What constitutes a human life? Do embryonic stem cells qualify? c) Should the government regulate embryonic stem cell research? d) Should frozen embryos from IVF be used for stem cell research? e) Should therapeutic cloning be used medically to obtain embryonic stem cells?

VIII. Vaccines A. New vaccines are being developed using new vaccine vectors and new delivery approaches, immunoenhancers, and nucleic acids. B. Vaccines are being developed against pneumonia, malaria, herpes virus, and others. C. New vaccines usually contain the antigen and not the organism. D. Small pieces of DNA from a microbe also induce an immune response, and are being studied to develop vaccines against malaria and HIV.

IX. Tissue Engineering A. Focuses on the development of substitutes for damaged tissues and organs. B. Combines biology and engineering to develop new tissue or the implantation of new cells. C. Occurs in the following way: 1. Tissue-inducing compounds, such as growth factors, serve as signals to stimulate the growth and development of new tissues. 2. The cells reside within a natural or synthetic extracellular matrix that can be incorporated into a patient’s tissue. Scaffolds or networks of polymers may serve as a substrate for cell growth and formation into tissues. 3. Isolated cells can be kept from host tissues to avoid rejection while they develop. 4. When implanted in the body, blood vessels grow and provide nutrients. D. Stem cells can potentially be used for tissue engineering. E. Research has focused on transplanting functional pancreatic islet cells coated with alginates to treat diabetes. The cells are implanted into animals, and are linked to the bloodstream by a tube that prevents antibodies and white blood cells from making contact with the implanted tissue. F. A biohybrid kidney was developed that maintains kidney function until an injured kidney recovers. The kidney was made of hollow tubes seeded with kidney stem cells that divided until they lined the tubes and became functional kidney cells.

X. Xenotransplantation A. A possible way to alleviate organ donor shortages is through organs from other animals, called “xenotransplantation. ” B. Tissue rejection, along with ethical and legal dilemmas, are major obstacles. C. Shield proteins such as membrane cofactor protein (MCP) and decay accelerating factor (DAF) are being researched because they can protect foreign tissue from attack by the human immune system, more specifically the complement system. D. Pigs are the best possible organ donor because their major organs are similar to those of humans in size and shape, and because few diseases are transferred from pigs to humans. E. Pigs have been produced with shield proteins and may reach clinical trials. F. In 1999, 160 people received pig cells without any adverse reactions.

XI. Drug Delivery A. Why Good Delivery Systems Are Needed. 1. Drugs are only useful if an effective method of delivery is available. 2. Aerosols are a possibility, since they are inhaled through the nose and pass into the lungs, where they are easily passed into the body through the many blood vessels. 3. Skin is also a candidate, because a transdermal patch along with an electric current can transfer large peptides that do not normally diffuse through the skin into the skin and then into the bloodstream. Biosensors. 1. Biological components such as a cell, antibody, or protein and are linked to a very small transducer. The transducer receives the signal and transforms it into an understandable form. 2. Transducers bind to the molecule, which produces an electrical or optical signal that can be detected. 3. Things that can be measured include blood components, environmental pollutants, toxins, and biological warfare agents. 4. Nanotechnology make biosensors so small that they can be easily concealed, or even placed into the body.

C. Biotech Revolution: Nanotechnology. 1. The study, manipulation, and manufacture of extremely small tools, structures, and machines at the molecular and atomic levels. 2. A field that involves engineers, physicists, chemists, and molecular biologists. 3. Possible applications include: a) b) c) d) e) Nanowires—very small wires to transit electricity. Nanobots—robots that assemble products. Nanomaterial production—create materials such as powders. Drug delivery—tiny containers may deliver drugs to the correct places. DNA computers—use DNA as hardware and software. 4. Biological molecules may provide frameworks instead of material like silicon, potentially using DNA to perform computing tasks such as mathematics. 5. Scientists have demonstrated that 1000 DNA molecules can solve, in four months, complex problems that would take a computer 100 years to solve.