Refresher Course in Life Sciences Academic Staff College (ASC), Osmania University (August 5 th -28 th , 2013) Group Project on GENE THERAPY -genes as the By By medicine Dr.A.Sab .Sabith itha a Rani ni Dr. r.C. Jyot othsn sna Lt.E.M.S .Sunith itha Dr.M.Madhavi Madhavi Dr.K. Shailaja laja Dr.Raf afat at Yasmee een
WHAT IS GENE THERAPY ? Definiton: an experimental technique for correcting defective genes that are responsible for disease development The most common form of gene therapy involves inserting a normal gene to replace an abnormal gene Other approaches used: Replacing a mutated gene that causes disease with a healthy copy of the gene. Inactivating, or “knocking out,” a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease.
Gene therapy is the insertion of genes into an individual cells and tissues to treat a disease in which a defective mutant allele is replaced with a functional one DNA is used as a therapeutic agent Genetic diseases, hematological disorders, acquired immunodeficiency syndromes, cancers are mainly treated
Researchers are studying gene therapy for a number of diseases, such as Severe combined immuno-deficiencies (SCID) Hemophilia Parkinson's disease Cancer HIV
Diseases for applying gene therapy Disease Defect Target cell Severe combined Adenosine deaminase 4 Bone marrow cells or immunodeficiency T-lymphocytes Hemophilia Factor VIII, Factor IX deficiency Liver, muscle. Cystic fibrosis Loss of CFTR gene Airspaces in the lung or globulin gene Hemoglobulinpathies Bone-marrow cells 1-antitrypsin deficiency 1-antitrypsin Lung or liver cells Cancer Many causes Many cell types Neurological diseases Parkinson’s, Alzheimers Direct injection into the brain Cardiovascular Restenosis, arteriosclerosis Vascular endothelium Infectious diseases AIDS, hepatitis B T cells, macrophages, Liver cirrhosis Fibrogenesis Hepatocyte growth factor MHC, 2-microglobulin Autoimmune disease Lupus, diabetes
GERM LINE GENE THERAPY • In germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are integrated into their genomes. • Result in permanent changes. • This would allow the therapy to be heritable and passed on to later generations. • Potential for offering a permanent therapeutic effect for all who inherit the target gene. • Possibility of eliminating some diseases from a particular family. • Also raises controversy: Some people view this type of therapy as unnatural, and liken it to "playing God” . Others have concerns about the technical aspects.
SOMATIC GENE THERAPY • The therapeutic genes are transferred into the somatic cells, or body, of a patient. • Affects only the targeted cells in the patient, and is not passed to future generations. • Short-lived because the cells of most tissues ultimately die and are replaced by new cells. • Appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases.
Types of somatic gene therapy
In vivo gene therapy 1. The genetic material is transferred directly into the body of the patient 2. More or less random process; small ability to control; less manipulations 3. Only available option for tissues that can not be grown in vitro; or if grown cells can not be transferred back
Ex vivo gene therapy 1. The genetic material is first transferred into the cells grown in vitro 2. Controlled process; transfected cells are selected and expanded; more manipulations 3. Cells are usually autologous; they are then returned back to the patient
Mec Mechan hanism ism of of Gen Gene T e Ther herapy Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by Vectors Two major methods of gene transfer Viral vectors • Retrovirus • Adenovirus • Adeno-associated virus • Herpes simplex virus Non-viral vectors • Naked DNA/Plasmid • Liposomes
Viral vectors • Virus replicate by inserting their DNA into a host cell as part of their replication cycle. • Gene therapy uses this by removing viral DNA and using the virus as a vehicle to deliver the therapeutic DNA. • Human gene therapy utilizes number of viruses like retrovirus, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus etc
Retr etrovir viruses uses • Retroviruses are RNA viruses which possess a reverse transcriptase function • Following infection (transduction) reverse transcriptase transcribes the viral RNA genome resulting CDNA copy and integrate into the human genome • DNA transfer is very efficient and stable, offering the possibility of a permanent cure for a disease. • Most promising vehicles for gene delivery and about 60% of clinical protocols utilize retroviral vectors. *The maximum size of DNA insert is 8kb *Can only transduce dividing cells, therefore limits potential target cells
Lentiv Lentivir iruses uses ( ( e.g. HIV) e.g. HIV) • Lentivirus are RNA virus, includes HIV virus. • These are complex retroviruses that infect macrophages and lymphocytes • Unlike retroviruses, lentiviruses are able to transduce nondividing cells and integrate into host cell chromosomes • Now considerable efforts are being devoted to making lentivirus vectors for gene therapy. *Maximum insert size 7-7.5 kb *May cause intentional mutagenesis
Adeno Adenovir viruses uses • Adenovirus are Double stranded DNA virus that cause respiratory, intestinal and eye infections in humans • Second most popular delivery system in gene therapy • These are Human viruses, infecting dividing and non-dividing cells • Large viruses and potential for accepting large insert size > 30 kb *Extensive unwanted immunological responses *Pre-existing host immunity
Adeno-As Adeno Associa sociated ted Vir iruses uses (AAVs) Group of small, single stranded DNA viruses Productive infection only with co-infection by helper virus, such as an adenovirus or herpes simplex virus. AAV vectors can accommodate inserts up to 4.5 kb, but have long-term gene expression Provide high degree of safety: because 96% of parental AAV genome is deleted with the gene of interest
applications are expected to be in delivering genes into neurons for the treatment of neurological diseases, such as Parkinson's disease and for. Her Herpes pes simple simplex x Vir iruse uses s (HSV) • Complex double stranded DNA vectors • Infects central nervous system (CNS) and can establish lifelong latent infections in neurons • Have comparatively large insert size capacity (>20 kb) but are nonintegrating and long-term expression of transferred genes is not possible • Used for delivering genes into neurons for the treatment of neurological diseases (Parkinson's disease and CNS tumors)
Lipo Liposome omes-Non Non vir iral al vect ector ors • Spherical vesicles composed of synthetic lipid bilayers which mimic the structure of biological membranes. • The DNA to be transferred is packaged in vitro with the liposomes and transferred to target tissue in vivo • L ipid coating allows the DNA to survive in vivo , bind to cells and endocytosed into the cells. • These are the popular vehicles for gene transfer • Unlike viral vectors, DNA/lipid complexes are easy to prepare and there is no limit to the size of DNA that is transferred. * Efficiency of gene transfer is low * Introduced DNA is not designed to integrate into chromosomal DNA.
Direct injection/particle bombardment • DNA can be injected directly with a syringe and needle into a specific tissues • Particle bombardment (‘gene gun’) : DNA is coated on to metal pellets and fired from a special gun into cells. • Successful gene transfer into number of different tissues can be obtained • Direct injection is simple and comparatively safe. However, there is poor efficiency of gene transfer and a low level of stable integration
1970s and earlier: • In 1972 Friedmann and Roblin authored a paper in Science titled "Gene therapy for human genetic disease?" .
First Approved Gene Therapy Mature T-cells GT September 14, 1990. Professor • Ashanti DeSilva; advanced stage of SCID; 4 yr old; William • Cynthia Cutshall January 31, 1991 French Anderson • Ashanthi De Silva - A rare genetic disease called severe combined immunodeficiency (SCID) • Defective adenosine deaminase gene results in deficiency of ADA protein • It plays important role in deamination reaction • Lack of healthy immune system
X-linked SCID (bubble disease) "bubble boy" disease, named after David Vetter, a Texan who lived out his 12 years in a plastic, germ-free bubble. More severe than ADA-SCID, as X-SCIDs have no B-, T-, NK cells Gene therapy trial for X-linked SCID successed in 2000; 8 of 10 patients significantly improved and live normal life.
Gene Therapy for Parkinson’s Disease In 2003 , University of California, Los Angeles research team inserted genes into the brain using liposomes • It is a significant achievement because viral vectors are too big to get across the blood – brain barrier. • This method has potential for treating Parkinson's diseaseI
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