Gene therapy
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This document discusses gene therapy, explaining the basics of genetics, types of human diseases, and the gene therapy techniques used to correct genetic disorders. It covers the mechanisms of gene therapy, including the use of viral and non-viral vectors, and various gene delivery methods and therapies aimed at treating conditions like cystic fibrosis and cancer. The importance of genetic research in understanding disease mechanisms and developing new treatment approaches is also emphasized.
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Gene therapy
- 1. Gene Therapy Correcting defective genes Dr. Mohit Kulmi
- 2. Basics of Genetics • Nucleotides are the alphabets of DNA. • Adenine (A), Thymine (T), Guanine (G) and Cytosine (C). • They always pair, A with T, and G with C. • These pairs are called "base pairs". • A human genome is made of 3,000 million base pairs, split into 46 chromosomes. • Almost every cell in our body contains a complete copy of our genome.
- 3. Continued… • A gene is the basic physical and functional unit of heredity. • Genes, are made up of DNA, and act as to make proteins. • Proteins carry out most of life’s function. • When altered causes dysfunction of a protein • In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases.
- 4. Continued… • The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes. • Only about 3% of the DNA actually codes for genes; the rest is often called "non-coding DNA" because its function is unknown. • The first complete human genome was only decoded in 2007.
- 5. Continued… • Every person has two copies of each gene, one inherited from each parent. • Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people. • Alleles are forms of the same gene with small differences in their sequence of DNA bases. • These small differences contribute to each person’s unique physical features.
- 7. Genetic Basis of Human Diseases Based on their genetic contribution, human diseases can be classified as • Monogenic, • Chromosomal, • Multifactorial. Monogenic diseases • are caused by alterations in a single gene, • and they segregate in families according to the traditional Mendelian principles of inheritance.
- 8. Continued… Chromosomal diseases as their name implies, are caused by alterations in chromosomes. • For instance, within an individual's genome, some chromosomes may be missing, • extra chromosome copies may be present, or • certain portions of chromosomes may be deleted or duplicated.
- 9. Continued… Multifactorial • The vast majority of human diseases can be categorized as multifactorial. • These conditions are also referred to as complex diseases, • They are responsible for most of the burden on our health care system.
- 10. Continued… • Examples of these conditions include • cardiovascular disease, • cancer, • diabetes, • a number of birth defects • and psychiatric disorders. • By definition, complex diseases are caused by variation in many genes, and they may or may not be influenced by environment.
- 11. Need for genetic Research • The goal of genetic research is to better understand the mechanisms of disease so that new treatment approaches and preventative measures can be proposed. • Now it is possible with the aid of latest technology to identify the genes responsible for disease and its exact location. • With this knowledge various therapeutic approaches can be undertaken to cure the disease.
- 12. Continued… • Any particular disease can be approached at the following levels: 1. At the level of genes encoding the defective protein, 2. At the level of that particular protein causing diseases, 3. And the secondary effects caused by the entry of defective protein into the cellular machinery.
- 13. What is Gene Therapy? • Gene therapy is the application of genetic principles in the treatment of human disease. • Gene therapy typically involves the insertion of a functioning gene into cells to correct a cellular dysfunction or to provide a new cellular function (Culver, 1994). • GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized to improve a genetic disorder.
- 14. Continued… For example, diseases such as • Cystic fibrosis, • Severe combined immunodeficiency syndrome, • Muscular dystrophy, • Hemophilia, and • many cancers result from the presence of defective genes. • Gene therapy can be used to correct or replace the defective genes responsible.
- 15. Continued… • Gene therapy is not a new idea. • In 1963, Joshua Lederberg wrote: • “The ultimate application of molecular biology would be the direct control of nucleotide sequences in human chromosomes,. . . . It will only be a matter of time . . . before polynucleotide sequences can be grafted by chemical procedures onto a virus DNA.”
- 16. Continued… • Less than 30 years later, the first clinical study using gene transfer was reported (Rosenberg et al., 1990). • Rosenberg and his colleagues used a retroviral vector to transfer the neomycin resistance marker gene into tumor- infiltrating lymphocytes obtained from patients with metastatic melanoma.
- 17. Variety of Gene Therapy Monogenic gene therapy • Provides genes to encode for the production of a specific protein • Cystic fibrosis, Muscular dystrophy, Sickle cell disease, Haemophilia, • SCID Suicide gene therapy • Provide ‘suicide’ genes to target cancer cells for destruction • Cancer
- 18. Continued… Antisense gene therapy • Provides a single stranded gene in an ’antisense’ (backward) orientation to block the production of harmful proteins • AIDS/HIV
- 19. Types of Gene Therapy Somatic gene therapy can be broadly split into two categories: Ex vivo: which means exterior (where cells are modified outside the body and then transplanted back in again). • Cells from the patient’s blood or bone marrow are removed and grown in the laboratory. • The cells are exposed to the virus that is carrying the desired gene.
- 20. Continued… • The virus enters the cells and inserts the desired gene into the cells’ DNA. • The cells grow in the laboratory and are then returned to the patient by injection into a vein. • This type of gene therapy is called ex vivo because the cells are treated outside the body.
- 22. In Vivo In vivo: which means interior (where genes are changed in cells still in the body). • During In vivo gene transfer, the genes are transferred directly into the tissue of the patient. • In vivo techniques usually utilize vectors. • Virus is the carrier of desired gene. • Virus is usually modified to disable its ability to cause disease.( E.g. deletion of env gene).
- 23. Continued… • Viral methods have proved to be the most efficient to date. • Many viral vectors can integrate the desired gene into the target cell’s genome. • Viruses are highly evolved natural vectors for the transfer of foreign genetic information into cells. • But to improve safety, they need to be replication defective.
- 24. Approaches to gene therapy Several approaches to gene therapy are being tested, including: - 1. Replacing a mutated gene that causes disease with a healthy copy of the gene. 2. Inactivating, or “knocking out,” a mutated gene that is functioning improperly. 3. Introducing a new gene into the body to help fight a disease.
- 25. Continued… Vector systems can be divided into: • Viral Vectors: Gene transfer mediated by viral vectors is referred to as transduction. • Non-viral Vectors: Gene transfer mediated by non-viral vectors is referred to as transfection.
- 26. General approaches used in gene therapy Non Viral Viral Based Naked/plasmid DNA Retrovirus Adenovirus Lipofection Adeno-associated Virus Gene Gun Lenti Virus Herpes simplex Virus
- 27. In vivo versus ex vivo • In vivo = delivery of genes takes place in the body • Ex vivo = delivery takes place out of the body, and then cells are placed back into the body
- 28. Viral Vectors Adenovirus Adenoviruses are: • medium-sized (90–100 nm), • Non-enveloped (naked), • Icosahedral viruses • Composed of a nucleocapsid and a double-stranded linear DNA genome. • There are over 51 different serotypes in humans, which are responsible for 5–10% of upper respiratory infections in children, and many infections in adults as well.
- 29. Continued… • The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material. • The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene.
- 30. Continued… • The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have that extra gene.
- 32. Adeno-associated Viruses • Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single stranded DNA. • These viruses can insert genetic material at a specific site on chromosome 19 with near 100% certainty. • They do not cause any known disease and doesn't trigger patient immune response.
- 33. Continued… • AAV has Low information capacity (it can carry small amount of DNA). • Gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. • Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases. • This is possible because AAV viruses can infect non-dividing (quiescent) cells, such as neurons.
- 34. Herpes Simplex Viruses • Herpes simplex viruses (HSV) belong to the subfamily of Alphaherpesvirinae. • HSV consists of a relatively large double-stranded DNA 150 kb in length, encased within an icosahedral capsid, which is wrapped in a lipid bilayer envelope. • The envelope is joined to the capsid by means of a tegument. This complete particle is known as the virion.
- 35. HSV
- 36. Continued… The unique features of HSV derived vectors are: • Very high transgenic capacity of the virus particle allowing to carry long sequences of foreign DNA, • the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, • and the ability of HSV vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia.
- 37. Lentiviruses • Lentivirus is a genus of the Retroviridae family, characterized by a long incubation period. • Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. • HIV, SIV, and FIV are all examples of lentiviruses. • Lentiviral infection have advantages over other gene therapy methods including high-efficiency infection of dividing and non-dividing cells, long-term stable expression of a transgene, and low immunogenicity.
- 38. Retroviruses • A retrovirus is any virus belonging to the viral family Retroviridae. • All The genetic material in retroviruses is in the form of RNA molecules. • When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes into the cell. • The retrovirus goes through reverse transcription using reverse transcriptase and RNA.
- 39. Continued… • the double stranded viral genome integrates into the human genome using integrase • integrase inserts the gene anywhere because it has no specific site. • May cause insertional mutagenesis • One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division) • vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety
- 40. Risk Factors with Viral Vectors • The concept of gene therapy seems straightforward, but this is clearly an oversimplification. • when viral vectors are used to carry genes into the body, they might infect healthy cells as well as cancer cells • new gene might be inserted in the wrong location in the DNA, possibly causing harmful mutations to the DNA or even cancer. • DNA could unintentionally be introduced into the patient’s reproductive cells. This could produce changes that may be passed on if a patient has children after treatment.
- 41. Continued… • The possibility that transferred genes could be overexpressed, leading to too much production of the missing protein as to be harmful, • the viral vector could cause an immune reaction. • The virus could be transmitted from the patient to other individuals or into the environment.
- 42. Non-Viral Vectors Non-viral methods present certain advantages over viral methods, • simple large scale production and low host immunogenicity. • Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; • however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.
- 43. Continued… Naked DNA • This is the simplest method of non-viral transfection. • Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; • however, the expression has been very low in comparison to other methods of transfection.
- 44. Continued… Electroporation: • Electroporation designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. • can be used to load cells with a variety of different molecules, like DNA transfer.
- 45. Lipoplexes and polyplexes • Lipoplexes and polyplexes, have the ability to protect the DNA from undesirable degradation during the transfection process. • Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome. • When the organized structure is complexed with DNA it is called a lipoplex. • Complexes of polymers with DNA are called polyplexes.
- 46. Continued… • Cheaper than viruses. • No immune response. • The most common use of lipoplexes has been to supply activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. • lipoplexes are useful in transfecting respiratory epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis. • 100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector.
- 49. Continued… Gene Gun Also known as a biolistic particle delivery system, • originally designed for plant transformation, • is a device for injecting cells with genetic information. • The payload is an elemental particle of a heavy metal coated with plasmid DNA. • This technique is often simply referred to as bioballistics or biolistics. • Gene guns have also been used to deliver DNA vaccines.
- 50. Continued… • Gene gun make use of the sticky nature of DNA or RNA when they adhere to biologically inert particles like metal atoms. • DNA is introduced by accelerating the DNA-Particle complex in partial vacuum. • Target tissue is placed in the acceleration path. • Gene gun is used to deliver a nucleic acid-based hepatitis B vaccine to both mice and humans, and is presently in clinical trials (Mumper, 2001).
- 52. Antisense Oligonucleotides Antisense Oligonucleotides : • The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. • Use of antisense oligonucleotides specific to the target gene to disrupt the transcription of the faulty gene. • Antisense Oligonucleotides are unmodified or chemically modified ssDNA, RNA or their analogs.
- 53. Continued… Some examples of Antisense Oligonucleotides: • Peptide nucleic acids (PNAs), • Locked nucleic acid (LNA), • Tricyclo-DNA (tcDNA), • siRNA.
- 54. Peptide Nucleic Acids(PNAs) • PNA was first introduced by Nielsen and coworkers in 1991. • They are electrostatically neutral molecules. • In PNAs the deoxyribose phosphate backbone is replaced by polyamide linkages, which is composed of repeating N-(2- aminoethyl)-glycine units. • PNAs can form very stable duplexes or triplexes with nucleic acids.
- 55. Locked Nucleic Acid (LNA) • LNA was synthesized by Jesper Wengel in 1998. • The ribose ring is connected by a methylene bridge (orange) between the 2’-O and 4’-C atoms thus “locking” the ribose ring in the ideal conformation for Watson-Crick binding.
- 56. Continued… • When incorporated into a DNA or RNA oligonucleotide, LNA makes the pairing with a complementary nucleotide strand more rapid and increases the stability of the resulting duplex. • As a result, LNA oligonucleotides exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. • LNA based hepatitis C drug called Miravirsen, targeting miR- 122, which is in Phase II clinical testing as of late 2010.
- 57. Tri-cyclo DNA (tc-DNA) • Much as LNA, tricyclo-DNA has been designed as a conformationally constrained oligonucleotide analogue. Chemically, tc-DNA deviates from natural DNA by three additional C-atoms between C(5’) and C(3’). •Increased stability towards nucleolytic degradation. •Increased RNA affinity.
- 58. RNA interference (RNAi) : • RNAi is an antisense mechanism that involves using small interfering RNA, or siRNA, to target a mRNA sequence. With siRNA, the cell utilizes a protein complex called RNA-induced silencing complex (RISC) to destroy the mRNA, thereby preventing the production of a disease-causing protein. • Applications of RNAi : • Cancer • HIV • Cardiovascular and Cerebrovascular Diseases • Neurodegenerative Disorders
- 60. Human artificial chromosome • A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. • Instead of 46 a cell can have 47 chromosomes with the 47th being very small, roughly 6-10 megabases in size, and able to carry newly introduced genes • researchers could integrate different genes that perform a variety of functions, including disease defense.
- 61. Continued… • The genetic material introduced by the vectors not only leads to different expression levels, but the inserts also disrupt the original genome. • HACs differ in this regard, as they are entirely separate chromosomes. This separation from existing genetic material assumes that no insertional mutants would arise. • stability and accuracy makes HACs preferable to other methods such as viral vectors
- 62. Continued… • I n 2010, a human artificial chromosome called 21HAC was reported. • 21HAC is based on a stripped copy of human chromosome 21 that is mitotically stable. • Using 21HAC, researchers were able to insert a herpes simplex virus- thymidine kinase coding gene into tumor cells. • This "suicide gene" is required to activate many antiviral medications. • These targeted tumor cells were successfully, and selectively, terminated by the antiviral drug ganciclovir. • This research opens a variety of opportunities for using HACs in gene therapy.
- 63. Severe Combined Immunodeficiency SCID is a primary immune deficiency • The defining characteristic is usually a severe defect in both the T- & B-lymphocyte systems. • Gene is located on chromosome 22 is inherited as an X-linked recessive disease, which for all practical purposes affects only boys. • results in the onset of one or more serious infections within the first few months of life. • SCID is caused by an Adenosine Deaminase Deficiency (ADA)
- 64. The Case of David Phillip Vetter • SCID is often called “bubble boy disease”. • SCID became widely known during the 1970′s and 80′s, when the world learned of David Vetter, • A boy with SCID, who lived for 12 years in a plastic, germ- free bubble.
- 65. Continued… • Immediately after being delivered David entered the plastic germ-free environment that would be his home for most of his life. • After being placed in the sterile chamber, David was touched only through special plastic gloves attached to the walls of the chamber. • David later received a bone marrow transplant from his sister, Katherine. Following that, David became ill.
- 66. Continued… • He died 15 days later, from Burkitt's lymphoma at the age of 12. • The autopsy revealed that the donor’s bone marrow contained traces of dormant Epstein-Barr virus which was undetectable in the pre-transplant screening. • Once transplanted, the virus spreaded and produced hundreds of cancerous tumors.
- 67. Continued… • Dr. W. French Anderson presides over the first experimental use of a gene therapy in 1990. The patient was a four-year old girl name Ashanti DeSilva. • She was born with SCID. • Dr. Anderson’s team drew blood from their patient, and replaced the defective gene with a functional variant. • The therapy partially restores Ashanti’s immune system.
- 68. Continued… • It temporarily stimulates production of the missing enzyme, but does not generate new cells with functional genes. • Ashanti continues to receive injections of corrected T-cells every two months. • She also takes doses of the enzyme itself, in the form of a drug called PEG-ADA. • She is able to lead a normal life.
- 69. Ornithine transcarbamylase deficiency • Gene therapy had been considered a possibility for curative treatment for OTC deficiency. • OTC deficiency is the most common urea cycle disorder in humans. • OTC deficiency is inherited in an X-linked recessive manner. • In severely affected individuals, ammonia concentrations increase rapidly causing ataxia, lethargy and death without rapid intervention.
- 70. Continued… • First case of death due to gene therapy was of Jesse Gelsinger. • He was suffering from OTC. • Jesse received gene therapy via adenovirus vector. • Soon after the therapy Jesse developed severe inflammatory reactions due to massive release of interleukin in response to adenovirus. • He died soon thereafter. • Currently, the only option for curing OTC deficiency is a liver transplant, which restores normal enzyme activity.
- 71. Few more examples… • Single gene disorder are the most suitable candidate for gene therapy. • Ex; • Cystic fibrosis: adenovirus administered via aerosol delivery into nasal passages and lungs. • Only transient expression observed because adenovirus does not integrate into genome like retroviruses. • Familial Hypercholesterolemia: Retroviral vector used to infect liver cells (~15% of patients liver) ex vivo. • Infused back into patient. • Improvement has been seen. • is used in many trials since then.
- 72. Continued… HIV: • University of Pennsylvania researchers have successfully genetically engineered the immune cells of 12 HIV positive patients to resist infection, and decreased the viral loads of some patients taken off the antiretroviral drug therapy (ADT) entirely. • By inducing the mutations, the scientists reduced the expression of CCR5 surface proteins. Without those, HIV cannot enter, rendering the patients’ cells resistant to infection. • The study also shows promise in the approach’s ability to suppress the virus.
- 73. How stem cells and gene therapy might work together 1. A sample of bone marrow is removed. 2. Stem cells are isolated and allowed to multiply in culture. 3. Cells are treated with a modified virus containing a therapeutic gene
- 74. 1. The virus is taken up by individual cells and the therapeutic gene goes into the cell's nucleus. 2. Treated ("corrected") cells are injected into the bloodstream. 3. Treated cells respond to injury signals from degenerating muscle or other tissues and migrate out of the bloodstream. 4. Treated cells patch damage and build healthy tissue.
- 75. Limitations of Gene Therapy Short Lived: • It is Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene. • Have to give multiple rounds of therapy. Gene delivery: • Dependence on cell cycle by some viral vectors (i.e. mitosis required) • Most viral vectors are unable to accommodate full length human genes containing all of their original regulatory sequences • Random integration can adversely affect expression
- 76. Continued… Duration of gene activity: • Non-integrating delivery will be transient (transient expression) • Integrated delivery will be stable Patient safety: • Immune hyper-responsiveness (hypersensitivity reactions directed against viral vector components or against transgenes expressed in treated cells). • Integration is not controlled oncogenes may be involved at insertion point, can lead to cancer. • Viruses may also cause disease once inside
- 77. Continued… Multigene Disorders: • Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because one need to introduce more than one gene • May induce a tumor if integrated in a tumor suppressor gene because of insertional mutagenesis. One problem with gene therapy is that one does not have control over where the gene will be inserted into the genome.
- 78. Continued… • The location of a gene in the genome is of importance for the degree of expression of the gene and for the regulation of the gene (the so-called "position effect"), and • thus the gene regulatory aspects are always uncertain after gene therapy . Expense: • Costly because of cell culturing needs involved in ex vivo techniques. • Virus cultures for in vivo delivery.
- 79. Recent Developments • Genes introduced into CNS using liposomes coated in polymer have the potential for treating Parkinson’s disease. • RNA interference or gene silencing to treat Huntington’s disease • Introduction of normal frataxin gene in animal models of friedreich’s ataxia with the help of AAV has shown promise. • Gene therapy leads to robust improvements in animal model of a fatal congenital pediatric disease known as X-linked myotubular myopathy.
- 80. Continued… • According to a recent study delivering a single injection of gene therapy to the spinal cord of rats following injury promotes the survival of nerve cells and improves hind limb function within weeks. • The effects of gene Cyclin A2 (CCna2) using gene therapy as a tool for cardiac regeneration, could lead to a viable treatment option for patients who suffer from myocardial infarction, or heart attack. • New gene therapy proves promising as hemophilia treatment.
- 81. Conclusion • As the science and technology will advance new therapeutic ways will always be discovered. • Gene therapy has a very wide application. • Almost every human disease has a genetic basis, -but with the current limitations of delivery vectors and the limited knowledge of human DNA, -it will take a long time to develop designer therapies for the patients. • By using gene therapy one day most of the diseases will be diagnosed before birth and cured. • Ultimately someday gene therapy will take human race to the pinnacle of evolutionary excellence.