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GENE THERAPY Dr. Walaa Wadie What is Gene Therapy? Using nucleic acids-based molecules (genes) as drugs. A key component is vehicles used to deliver therapy to cells &/or organs. Gene-based molecules + vehicle = gene therapy Focus & goal of gene therapy Inherited disorders: (e.g. cystic fibrosis, hemophilia, adenosine deaminase deficiency). To correct genetic defects permanently and thereby restore normal cellular function. Acquired diseases: (such as cancer). To cure disease by targeting pathogenic processes. Types of gene therapy Somatic gene therapy − Exogenous genes (= transgenes) are transferred into somatic cell of the recipient. − Not affect future generation. Germ-line gene therapy − Transgenes are transferred into germ cells (sperm or eggs) of the recipient. − Affect future generation. Gene Therapy Versus Conventional Therapy Gene Therapy Conventional Therapy Materials DNA, RNA; etc. Chemicals, Peptide, Proteins. Mechanisms Usually cure the causes of the diseases Usually relieve the symptoms or signs Duration of Effect Can be permanent and also can be passed down to next generation in germ-line gene therapy. Usually stop the effect once stop taking it. Ethics Major Issues (germ-line gene therapy) Usually Not General guidelines in gene therapy Disease not successfully treated by current therapies. Genetic basis of disease must be determined. Pathophysiology of disease must be known. Magnitude & duration of gene expression must be estimated. Tools for detection of expression must be in hand. Methods of Gene Transfer In vivo: Direct administration of the gene therapy formulation to patients. Ex vivo: Transfection of cells in tissue culture by gene therapy followed by administration of the transfected material into the patient. Cells must be capable of removal, survival outside the body & re-implantation e.g. hepatocytes, skin fibroblasts, lymphocytes, etc. Gene Delivery Systems Non-viral vectors: Naked DNA, DNA–polymer conjugates & liposomes. Viral vectors: DNA & RNA vectors Retrovirus, Adenovirus, simplex virus. Adeno-associated virus, Herpes Gene Delivery Systems 1- Non-Viral Vectors Naked DNA (can be administered by direct injection of the naked DNA into some tissues, especially muscle, usually used in vitro and by electroporation) Adv: Ease of production. Safety. No DNA size limitation. Disadv: ↓ Efficiency. Transient expression. Gene Delivery Systems Physical methods to enhance delivery: Electroporation is a method that uses short pulses of high voltage to carry DNA across the cell membrane. Gene Gun Plasmid DNA was coated onto 1-3 µm gold or tungsten particles and after that ‘fired’ at the tissues via electrical or gas pulse acceleration Sonoporation uses ultrasonic frequencies to deliver DNA into cells Gene Delivery Systems Electroporation Gene Delivery Systems Chemical methods to enhance delivery: a. Liposomes Lipid molecular aggregates that can carry naked DNA (lipoplex). Adv: Ease of production. Low immune reactions. No DNA size limitation. Disadv: ↓ Efficiency. Transient expression. Gene Delivery Systems b. DNA polymer conjugate (polyplex) - - DNA + +++++ Ligand Polycation e.g. polyethylenimine DNA Complex Gene Delivery Systems Non-viral vectors DNA Lipoplex Polyplex Production Easy Easy Easy Capacity unlimited unlimited unlimited Transfection Efficiency low low low Integration Non Non Non Expression Transient Transient Transient Immune Reactions Gene Delivery Systems 2- Viral Vectors Design focuses on efficacy & biosafety. Rendering virus vector harmless: Remove disease-causing genes and replace by therapeutic gene (gene of interest). The viral genes that control delivery mechanisms are retained. Gene Delivery Systems Target cell Virus may be: Vector uncoating Viral vector Expression Integration & Expression Therapeutic mRNA & protein • Non-Integrating viruses (transient expression) – Adenovirus – Herpes Simplex Virus • Integrating viruses (stable expression) – Adeno-associated virus – Retrovirus – Lentivirus Gene Delivery Systems A. DNA-Viral Vectors Adenovirus: Adv: Ease of production. Efficient DNA transfer (High transfection efficiency) Can infect dividing and non-dividing cells. Disadv: Transient expression (< 10 days)(viral DNA does not integrate) Immunogenicity: life-threatening immune response. In vivo delivery hampered by host immune response (prevents any subsequent transfection even if a second injection of the recombinant adenovirus is given) Gene Delivery Systems Adeno-associated Vectors (AAV): Adv: ↓ Immunogenicity Transduce dividing and non-dividing cells. Prolonged expression (up to 9 months). Disadv: Limited DNA capacity. Difficult production. Gene Delivery Systems Herpes Viral Vectors: Adv: Targets CNS (brain cancer). ↓ Immunogenicity. Disadv: Difficult to manufacture. Transient expression. Low transduction efficiency. Gene Delivery Systems B- RNA-Virus Vectors Retroviruses: Adv: • Low immune reactions. • Easily manufactured. • Efficiently transferred. • Stable expression. Disadv: • Targets dividing cells only. • Small DNA capacity. • Risk of insertional mutagenesis. The integrase enzyme can insert the genetic material of the virus into any arbitrary position in the host chromosome (randam DNA insertion). If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e. cancer) can occur. Gene Delivery Systems Lentivirus vectors (subclass of retrovirus) Infect dividing and non-dividing cells. Easily produced. Low cellular immune response. Sustained expression over six months Viral vectors DNA-viral vector RNA-viral vector Adenovirus Production Capacity Transfection Integration Expression Easy limited Efficient Non Transient Immune Response High Target AAV Lentivirus Herpes Retrovirus (subclass of Simplex V retrovirus) difficult difficult limited limited Low Efficient Yes Non Stable Transient Easy limited Efficient Yes Stable Easy limited Efficient Yes Stable low low low low Dividing & nondividing cells CNS Dividing cells Dividing & nondividing cells (brain cancer) Risk of insertional mutagenesis Gene Delivery Systems N.B.: Generally, viral vector system show higher gene transfer efficiency than non-viral gene carrier system, but viral systems have potential risk of immunogenecity and cancer formation. Gene Therapy Approaches N.B.: Diseases at a genetic level result from: Loss of expression of a gene. Mutation of a gene. Elevated expression of a gene. Expression of pathogenic viral or foreign gene. Gene Therapy Approaches • Replacing missing or mutated gene with a healthy copy of gene. Scientists focused on diseases caused by single-gene defects (monogenic disease) e.g. Cystic fibrosis, Haemophilia, SCID • Gene silencing: • suppress expression of undesired gene. • Include: RNA interference, Antisense therapy. • e.g. AIDS/HIV • Suicide gene therapy: • Introducing a new gene “suicide gene” into the body to help fight a disease. • e.g. Cancer Gene Therapy Approaches Replacing missing or mutated gene (DNA + vector) DNA Transcription Translation Gene Therapy Approaches ………Replacing missing or mutated gene The produced protein may: function intracellularly. or secreted into circulation. Evaluation of successful gene transfer (transfection) is done by: • • • Measuring mRNA (transcription). Measuring protein (translation). Measuring function of target cells. Gene Therapy Approaches Gene silencing: Nucleic acids can be delivered to interrupt specific mRNA translation & protein synthesis. (So prevent mutated or overactive genes from directing protein synthesis). Two mechanisms exist to destroying targeted mRNA: a. Antisense therapy. b. RNA interference. silence genes by Gene Therapy Approaches ………Gene silencing a. Antisense Therapy mRNA Transcription Translation DNA Protein Using antisense oligonucleotides (ASON) to ↓ expression of a target gene by binding to mRNA. ASON mRNA Transcription DNA Translation Gene Therapy Approaches ………Gene silencing ….Antisense Therapy Antisense oligonucleotides (ASON): • are sequences of 17-30 bases of single-stranded DNA that are complementary to a chosen sequence of target mRNA. • are of short length to facilitate cell internalization & ↑ hybridization efficiency. Mechanism of action: ASON bind to target mRNA sequence DNA-RNA duplex activation of ribonuclease H (RNase-H). RNase-H ASON-mRNA free ASON +degraded mRNA. Net result → ↓ mRNA translation & protein synthesis. Gene Therapy Approaches ………Gene silencing ….Antisense Therapy Gene Therapy Approaches ………Gene silencing b. RNA interference: Mechanism of action: Large double strand RNA (dsRNA) sequence designed to target endogenous mRNA enter the cell. dsRNA is cut by DICER enzyme to short interfering RNA (siRNA). siRNA bind to a group of proteins called RNA-induced silencing complex (RISC). Gene Therapy Approaches ………Gene silencing …. RNA interference RISC performs 2 imp jobs: – activates unwinding of siRNA to single strand RNA that binds to target mRNA molecule. – cuts mRNA in the regions paired with single strand RNA. Gene Therapy Approaches ………Gene silencing …. RNA interference + Large dsRNA DICER siRNA RISC Unwind siRNA Single-strand RNA + RISC mRNA degradation Target mRNA Gene Therapy Approaches ………Gene silencing …. RNA interference Gene Therapy Approaches ………Gene silencing Gene Therapy Approaches Suicide gene therapy Introducing a new gene “suicide gene” into the body to help fight a disease. This approach is used in cancer gene therapy. 1. 2. Tumor cells are transfected with a gene coding for an enzyme such as herpes simplex virus-1 thymidine kinas. Systemic administration of ganciclovir. ganciclovir (nontoxic) --thymidine kinase its active cytotoxic form death of tumor cells. Therefore, cancer cells become more vulnerable, more sensitive to chemotherapy Gene Therapy Approaches ……… Suicide gene therapy Applications of Gene Therapy Obstacles facing gene therapy: Immunogenicity. ↓ Efficiency. Transient transgene expression. Mutagenicity or oncogenesis Ethical factors (germ-line gene therapy): DNA could accidentally be introduced into reproductive cells Gene product toxicity: expression is higher than normal endogenous levels & concentrated within localized population of cells. Applications of Gene Therapy Gene Therapy Human Application Considerations Specificity Effective Safety Only desired cells or tissue The delivery efficiency dependent on the disease requirements Biocompatible Non-cytotoxicity Non-immunogenecity Non-inflammation Non-Tumor Generation Applications of Gene Therapy (AIDS) Cancer Applications of Gene Therapy Cancers: 1) Addition of a tumor suppressor gene (genes encoding P53). 2) Expression of immunomodulating expressing IL-2). gene (genes 3) Deliver genes expressing co-stimulatory molecules necessary for activation of T-lymphocytes. 4) Use ASON to turn-off expression of an oncogene (bcl-2 oncogene). 5) Inhibition of tumor angiogenesis (suppress angiogenic growth factor VEGF). 6) Transfer of a gene leading to tumor-specific cell killing (suicide gene therapy). Applications of Gene Therapy Infectious diseases: HIV/AIDS produce HIV-infected cells that express thymidine kinase that activate non-toxic prodrug ganciclovir into cytotoxic one (suicide gene therapy). – Trials to – Decrease HIV replication by modification of CD4 T cells ex vivo to express proteins that interfere with HIV transcription. Cytomegalovirus retinitis Fomivirsen (marketed as Vitravene)- 21-base pair ASON- 1st ASON agent approved by FDA – used for CMV retinitis in HIV patients. Applications of Gene Therapy Monogenic diseases Disease Defect Target Cells Severe combined Adenosine deaminase Bone-marrow immunodeficiency (ADA) deficiency in 25% cells or T of SCID patients lymphocytes (SCID) Cystic fibrosis (CF) Defect in Cystic fibrosis transmembrane conductance regulator Airways in the (CFTR) lungs Faulty transport of Cl- in lung epithelium Hemophilia -- A (80%) Factor VIII deficiency -- B (20%) Factor IX deficiency Liver, muscle, fibroblasts or bone marrow cells Applications of Gene Therapy Adenosine deaminase deficiency Severe combined immunodeficency syndrome (SCID) (Patients cannot withstand infection die if untreated) Applications of Gene Therapy Progress in SCID treatment: The baby is kept in a bubble-like structure (isolated in a germfree environment). David Vetter ‘Bubble boy’ (1971 - 1984). Bone marrow transplantation: the most common However, a proper 'bone marrow match' is necessary for transplantation. Gene therapy: () - 1st clinical gene therapy trial (1990): T cells were removed from body → ADA gene was inserted into these T cells → again injected into body → normal immune system. Only worked for a few months and therefore process (replacement gene therapy of T cells) should be repeated. -Recently, Strimvelis is the first ex-vivo stem cell gene therapy. Applications of Gene Therapy Applications of Gene Therapy Cystic fibrosis Applications of Gene Therapy Gene therapy in CF usually in form of aerosol spray Insert transgene into liposome or viral vector Applications of Gene Therapy Gene therapy in hemophilia Applications of Gene Therapy Multifactorial diseases: Delivery of genes coding for angiogenic growth factors e.g. VEGF ➙ stimulate vascular proliferation in coronary artery disease (CAD) & peripheral vascular diseases. Inflammatory diseases (RA, IBD, asthma): • Delivery of genes encoded with immuno-modulatory or anti-inflammatory cytokines. • ASON to silence pro-inflammatory cytokines. Thank you