Gene Therapy

1. Gene Therapy The Forefront of Medicine

2. Group Members • Maja Udovcic – Background and Introduction • Matthew Lee – Scientific Considerations • Vanessa Crawford – Social Considerations • Sean Park – Ethical Considerations • Emily Edwards – Case Studies

3. Gene Therapy - Background • 1865 - Mendel's experiments described the laws of heredity, and that features are inherited by a defined and predictable mechanism • 1940s - Avery and colleagues identified carrier of genetic information, demonstrated that the information is encoded by DNA Gregor Mendel’s Heredity Experiment

4. Gene Therapy - Background • 1953 –Watson and Crick proposed that DNA is a double helix, suggesting how this structure could be used to replicate and inherit genetic information • 1961 –Nirenberg deciphered triplets in the genetic code • 1978 – Arber, Nathans and Smith discovered restriction enzymes and applied it to problems of molecular genetics James Watson and Francis Crick

5. Gene Therapy - Background • 1990 - The first gene therapy journal published, Human Gene Therapy • 1990 - The first approved gene therapy clinical trial took place when Ashanthi DeSilva, a 4 year old girl with ADA-deficient Severe Combined Immunodeficiency, was given her own T cells engineered with a retroviral vector carrying a normal ADA gene • 2000 - The first gene therapy cure was reported when Alain Fischer (Paris) succeeded in totally correcting children with SCID-X1, or “bubble boy” syndrome “Bubble Boy”

6. Gene Therapy - Background

7. What Are Genes? • Genes are carried on chromosomes and are the basic physical and functional units of heredity • Genes are specific sequences of bases that encode instructions on how to make proteins • When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders result

8. What Are Genetic Disorders? • Genetic disorder is a disease caused by a "variation" or "mutation“ of a gene. • Genetic disorders can be passed on to family members who inherit the genetic abnormality. • A small number of rare disorders are caused by a mistake in a single gene. • Most disorders involving genetic factors, such as heart disease and most cancers, arise from a interplay of multiple genetic changes and environmental factors.

9. What Are Genetic Disorders? • Thee categories of genetic disorders: • Single gene disorders caused by a mistake in a single gene. Sickle cell, cystic fibrosis and Tay-Sachs disease are examples. • Chromosome disorders caused by an excess or deficiency of the genes. Down syndrome is caused by an extra copy of a chromosome, but no individual gene on the chromosome is abnormal. • Multifactorial inheritance disorders caused by a combination of small variations in genes. Heart disease, most cancers and Alzheimer's disease are examples. Sickle Cell Disorder

10. What is Gene Therapy? • Researchers may use one of several approaches for correcting faulty genes: • A normal gene may be inserted into a location within the genome to replace a nonfunctional gene. Most common approach. • An abnormal gene could be swapped for a normal gene through homologous recombination. • An abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. • The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

11. How Does Gene Therapy Work? • In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. • A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. • The most common vector is a virus that has been genetically altered to carry normal human DNA. • Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. • Scientists manipulate the virus genome to remove disease-causing genes and insert therapeutic ones. • Target cells, such as the patient's liver or lung cells, are infected with the viral vector.

12. How Does Gene Therapy Work? • Non-viral options: • Direct introduction of therapeutic DNA into target cells. Can be used only with certain tissues and requires large amounts of DNA. • An artificial lipid sphere with an aqueous core, called a liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane.

13. Problems With Gene Therapy? • Short-lived nature of gene therapy- patients will have to undergo multiple rounds of gene therapy. • Immune response- risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. • Problems with viral vectors- viruses, the carrier of choice, present potential problems to the patient, like toxicity, immune and inflammatory responses, and gene control and targeting. • Multi-gene disorders- most common disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis and diabetes, are caused by the combined effects of variations in many genes.

14. Is Gene Therapy Ethical? • Questions we will consider: • What is normal and what is a disability or disorder, and who decides? • Who will have access to your genetic information? • Is somatic gene therapy (done in the adult cells of people known to have the disease) more or less ethical than germline gene therapy (done in egg and sperm cells and prevents the trait from being passed on to further generations)? • Preliminary attempts at gene therapy are expensive. Who will have access to these therapies? Who will pay for their use?

15. Gene Therapy: A Scientific Perspective Gene Therapy has been defined as: nucleic-acid based treatment, or transfer of DNA/RNA to somatic target cells in the intention to treat serious illness’ (1). In somatic gene therapy, new genes are introduced to the body In germ line therapy, the human germ line is modified, conferring heritable modifications to the offspring However, germ line therapy is not permitted in any county, on the basis that it is unethical Essential to the progression of gene therapy is a comprehensive understanding of the human genome and various genetic diseases

16. Types of Gene Therapy • Prominent forms include postnatal gene delivery via viral vectors for insertion within the genome, imparting expression of the newly incorporated gene, and so-called “gain of function” • RNA interference, or RNAi, borrows from the principals of naturally occurring process within biological systems, used to affect relative levels of expression of certain genes. • Present research and ongoing efforts are also being made in the development of human prenatal gene therapy

17. In vivo gene therapy: delivery of new genetic material directly to target cells within the body • The challenge lies in ensuring the specificity and in reaching the correct target cells within the body • Ex vivo therapy: target cells are removed from the body and then genetically modified • The cells are then returned to the body after selection and amplification • This is a safe method but dependent on the type of cells being targeted

18. Postnatal Gene Therapy • Purpose: Correction of the deleterious effects of genetic disease via long term integration of gene sequences into a patient’s genome • This property makes the use of retroviral vectors particularly attractive when considering effective gene delivery to correct inherited monogenetic disorders

19. Types of Postnatal Gene Therapy • Gene replacement: non-functional or defective gene is replaced by a new, functional copy of the gene • Can be accomplished by homologous recombination, although efficiency is low • Gene addition: introduction of a gene that is able to produce a protein not normally expressed in the cell • i.e. Introduction of a so-called “suicide gene” into cancer cells

20. Vectors • Vectors are carrier molecules which are employed to enhance gene transfer efficiency in gene therapy • In optimizing a particular vector, one must consider: • Host immune response • Must target specific tissues for long term gene expresssion • Regulation of the gene after insertion • Both viral and non-viral vectors have been used, though non-viral have a decreased transfer efficiency

21. Gene Therapy Progress and Prospects Fetal gene Therapy: Also known as prenatal or in utero gene therapy Targets genetic diseases which require lifelong correction The concept of fetal gene therapy is based on the following aims: • avoiding early-onset manifestation of life-threatening genetic conditions • achieving permanent correction of such diseases by stable transduction of relevant fetal progenitor cell populations • Avoiding immune reactions against the therapeutic vector and transgene by induction of tolerance.

22. First proofs of principle for therapeutic in utero gene application • First successful therapeutic application of gene transfer in utero was carried out in 2003 by Seppen et al. • This was achieved by direct injection of a lentiviral vector expressing the human bilirubin UDP-glucuronyltransferase (UGT1A1) gene under control of the phosphoglycerate kinase promoter into the liver of Gunn rat fetuses.

23. Successful Therapeutic Applications

24. Benefits of prenatal gene therapy • Provides early phenotypic correction, reducing or avoiding otherwise devastating effects of genetic disease • Demonstration of long-term postnatal therapeutic protein production • Tolerance to the transgenic protein can be induced by in utero expression

25. “Although fetal gene therapy will not replace postnatal gene therapy, it is essentially a preventive approach to the management of otherwise predominantly incurable diseases and would therefore – if successful and safe – be most effectively conducted in conjunction with prenatal screening programmes.”

26. Progress in Prenatal Gene Therapy • Disparity between species must be taken into account when considering administration of human fetal gene therapy • Minimally invasive methods of ultrasound guided gene delivery are being devised in large animal models

27. Case Study: More to Come • First great testament to the power of gene therapy was in response to X-linked severe combined immunodeficiency disease (SCID) • Attempts to find alternative treatment options for this monogenetic disease led to one of the first great successes in gene therapy • In a landmark clinical trial, Alain Fischer’s group successfully treated children with X-linked SCID • However, the fickle finger of fate had more in store…to be continued…

28. RNA Interference • RNA interference, also known as RNAi presents a new approach to gene therapy by targeting specific genes and down-regulating gene expression • One of the most potent forms of RNAi is small interfering RNA, or siRNA • Small fragments of double stranded RNA, specific for a particular gene target, are introduced to the cell • Specific hybridization between the naturally occurring transcript and the induced siRNA (antisense portion) instigates the destruction of the message. • This form of RNAi acts directly on the transcriptional level of gene expression. • Therapeutically speaking, siRNA efficacy would be determined by percent knock-down (gene is still present, some product is still made). • Also, this method is transient, requiring readministration within the system.

29. Mechanism of RNAi

30. Molecular Biology Fun! • http://www.nature.com/focus/rnai/animations/rnai_revised_320x180.mov

31. RNAi Fun • Treatment-related activity of most small molecule drugs is to inhibit the function of its target molecule in as specific a manner as possible. • The specific manner in which RNAi functions serves as a major benefit to possible therapeutic applications… • But can its administration be optimized in terms of specificity and efficiency in in vivo models? • The principal issue in turning RNAi from an effective functional genomics tool into a therapy remains one of delivery. RNAi primarily acts within the cytoplasmic compartment, which is easier to access using nonviral methods than the nucleus, but ensuring efficient uptake and long-term stability in vivo in disease relevant tissues is still likely to be difficult.

32. “The principal issue in turning RNAi from an effective functional genomics tool into a therapy remains one of delivery. RNAi primarily acts within the cytoplasmic compartment, which is easier to access using nonviral methods than the nucleus, but ensuring efficient uptake and long-term stability in vivo in disease relevant tissues is still likely to be difficult.” –NJ Kaplen

33. Adverse Effects of Gene Therapy • Vector induced oncogenesis • Germline transfer of transgenic DNA sequences • Developmental aberrations caused by expression of the transgenic proteins and vector induced oncogenesis • Without proper specificity, delivery to the right cell type in the right organ, at the right time, there could be detrimental immunological effects.

34. The Ethics and Social Concerns Surrounding Gene Therapy

35. Ethics vs. Morality • Ethics: normative, seeks to objectively prescribe a standard for conduct • Morality: doctrine or system of conduct, implies conformity to established sanctioned codes, relates to right or wrong. Ex: the Church’s stance on gene therapy • “Ethicists seek to understand the basic principles underlying a subject from an unbiased point of view, while moralists seek to help people become better.” • We often confuse gene therapy morals with ethics.

36. The Discovery of Deoxyribonucleic Acid (DNA) • Over 40 years ago, Francis Crick and James Watson discovered the alpha-helical structure of DNA, the fundamental building block for gene therapy. • Francis Crick: “We used to think that our fate was in our stars. Now we know that, in large measure, our fate is in our genes.”

37. Crick’s Reductionist Approach • Advocates of gene therapy, including Crick, hold a firm reductionist approach - that all life can be reducible into simpler, more fundamental things. • Example: biology can be reduced to chemistry, and chemistry can be reduced to physics • Can all life be explained this way?

38. Problems With Scientific Reductionism • Epigenetics - some gene regulatory information not expressed in DNA sequences are transmitted from one generation to the next • Twin studies… • Anthropic Principle: “all life is fine-tuned” • the scope of science is limited by the laws of physics according to reductionism. • How do you explain consciousness?

39. History of Gene Therapy Ethics • 19th Century Biologist August Weismann establishes the “Weismann Barrier” • 1974: NIH regulates recombinant DNA research. Recombinant DNA Advisory Committee (RAC) to the NIH Director was created. • 1984: RAC creates new group, Human Gene Therapy Working Group (later called Human Gene Therapy Subcommittee, or HGTS) • 1984: U.S. Office of Technology Assessment (OTA) publishes background paper Human Gene Therapy • 1985: RAC Subcommittee prepares “Points to Consider” document for public presentation • 1992: NIH Director Dr. Bernadine Healy approves compassionate use exemption of gene therapy for critically ill patient • 1998: HUGO Ethics Committee makes statement on gene therapy ethics. • 1999: first death by gene therapy, Jesse Gelsinger reported in Nature. United States Senate hearings, NIH, and FDA consider the adverse effects of gene therapy.

40. HUGO Ethics Committee Statement on Gene Therapy Research - 1999 • Germ-line cell gene therapy avoided, only confronts somatic cell therapy • Main Objectives of Statement: • respond to public concerns about ethical conduct, quality, and safety of somatic gene therapy research • make distinction between somatic therapy from germ-line therapy • encourage adoption of international guidelines • propose framework for public accountability

41. HUGO Ethics Committee Statement on Gene Therapy Research (continued) • Common principles previously established by HUGO in 1996 Statement of the Principled Conduct of Genetics Research: • Recognition that the human genome is part of the common heritage of humanity • Adherence to international norms of human rights • Respect for the values, traditions, culture, and integrity of participants • Acceptance and upholding of human dignity and freedom

42. HUGO Ethics Committee Statement on Gene Therapy Research (continued) • Recommendations by HUGO Committee: • public oversight and ongoing review of research • countries need national ethics bodies that mandate somatic gene therapy • researchers and governments must respond to public concerns about the benefits, risks and ethical conduct of research • all research conducted must abide by stringent quality and safety controls and be in conformity with international norms

43. HUGO Ethics Committee Statement on Gene Therapy Research (continued...) • Recommendations by HUGO Committee (continued): • material conflicts of interest must be identified, declared, and addressed in the most transparent way possible • researchers and media reporting on gene therapy development must do so in an informative and responsible manner • widespread discussion on the appropriateness of possible future gene therapy technology using germ-line cells • It is crucial that the media presents gene therapy responsibly. After all, we are educating an uninformed, moral-driven public.

44. Problems With the HUGO Committee Statement • Why avoid the controversy surrounding germ-line cell therapy? • Lacks descriptive language, very vague. • What kind of material conflicts of interest exist today? • How will the general public be granted oversight over gene therapy development? Federal or state law?

45. Is There Legislation in Place to Protect Your Genetic Information? • There is No Federal policy in place to protect your genetic information unless you work for the Federal government. • There are however, some state policies in place… • http://www.ncsl.org/programs/health/genetics/prt.htm

46. Alabama Connecticut Idaho Indiana Iowa Kansas Kentucky Maine Minnesota Mississippi Montana North Carolina Ohio Oklahoma Pennsylvania Tennessee West Virginia Wisconsin Wyoming = 19 States States Without Legislation Protecting Genetic Information

47. 4 Ethical Yardsticks: Review • Nonmaleficence: Do no harm, avoiding the causation of harm. • Beneficence: Do good. • Respect for autonomy: Respect for the fundamental self-worth, dignity,and decision-making capacity of individuals. • Justice: Distribute benefits, risks and costs fairly.

48. Genetic-ism • Will parents who choose to have babies naturally raise children who will be ostracized in school? • Will a new generation of “perfect” people disrespect and disregard their elders and ancestors?

49. Geico Caveman Commercial

50. Genetic-ism • Where is the line that separates treatment and enhancement? • What will medical insurance cover? • Could this lead to the creation of a new form of “untouchables?”