Gene therapy

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Gene therapy:

Gene therapy Dr. Mayur M. Maybhate JR II Dept. of Pharmacology IGGMC, Nagpur.

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Introduction Types of gene therapy Methods of gene therapy Current applications Problems with gene therapy Conclusion


Introduction “No two individuals are same in this world, not even identical twins !!!” Large variation in phenotypic characters Genetic constitution decided at conception and it is fixed…..But….. Nothing is permanent… nothing is impossible !!!

What are genes ???:

What are genes ??? biological unit of heredity. Determine obvious traits, such as hair and eye color, as well as more subtle characteristics such as the ability of the blood to carry oxygen.

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A gene is a part of DNA molecule, and humans have about 30,000 genes. Genes carry ‘instructions’ that allow the cells to produce specific proteins such as enzymes DNA RNA Proteins

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Only certain genes in a cell are active at any given moment. As cell mature, many genes become permanently inactive. The pattern of active and inactive genes in a cell and the resulting protein composition determine what kind of cell it is and what it can do and cannot do. Flaws in genes can result in disease

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Mutation in genes Changes in codon sequence Altered transcription and translation Defective protein synthesis Disease

What is gene therapy ?:

What is gene therapy ? An experimental treatment that involves introducing genetic material (DNA or RNA) into a person’s cells to fight disease A novel approach to treat, cure, or ultimately prevent disease by changing the expression of a person’s genes

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4 approaches to correct defective gene - normal gene may be inserted into nonspecific location within genome to replace defective one - Abnormal gene can be swapped for normal gene through homologous recombination - Abnormal gene could be repaired through reverse mutation - regulation of particular gene could be altered

Father of gene therapy:

Father of gene therapy

The beginning !!!:

The beginning !!! In 1980s, advances in molecular biology enabled human gene to be sequenced and cloned Lot of diseases due to single gene defects such as cystic fibrosis, hemophilia, muscular dystrophy, sickle cell disease and many more…. Alteration of defective gene can cure disease ???

Types of gene therapy:

Types of gene therapy Germ line gene therapy Somatic gene therapy

Germ line gene therapy:

Germ line gene therapy Germ cells i.e. sperms or eggs are modified by introduction of functional genes into their genomes Change would be heritable to later generations, hence can remove the disease from family tree forever !!! Difficult technically, controversial ethically !

Somatic gene therapy:

Somatic gene therapy Therapeutic genes transferred into somatic cells of patient Beneficial effect will restrict for patient only, will not be inherited 2 types - Ex vivo - In vivo

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Ex vivo gene transfer - Few defective cells removed from patient and grown in the laboratory. - The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells and inserts the desired gene into the cell's DNA. - The cells grow in the laboratory and then returned to the patient by injection into a vein.

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In vivo gene transfer - The vectors carrying desired gene injected directly into the body.

Methods of gene delivery:

Methods of gene delivery

Why viruses ???:

Why viruses ??? High concentration of virus allowing many cells to be infected or transduced Convenience and reproducibility of production Ability to transduce dividing and non-dividing cells

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Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episome Ability to target the desired type of cell

Mechanism of viral gene delivery:

Mechanism of viral gene delivery viruses genetically altered to carry normal human DNA Target cell such as the patient’s liver or lung cells are infected with the viral vector. The viral vector then unload its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from therapeutic gene restores the target cell to a normal state.


Retroviruses Genetic material in form of RNA, while of host, it is DNA RNA molecule must produce a DNA copy before it can be considered part of the genetic material of the host cell Equipped with enzyme reverse transcriptase and integrase host cell now contains a new gene, multiplies, restores function



Disadvantages of retroviruses:

Disadvantages of retroviruses can only invade cells that devide often, therefore blood cells, skin cells and many other tissues can not be invaded by this vector. Do not insert their genetic material in any specific places, but in the middle of an important gene. The important gene could become defective, stop functioning and could do more harm than good……. ( insertional mutagenesis ) e.g. leukemia in 10 patients Immune rejection


Adenovirus Genetic material in the form of DNA (common pathogen) When these viruses infect a host, they introduce their DNA molecule into the host but not incorporated into the host genetic material . These extra genes are not replicated, so when the host undergo cell division, the descendants of the cell will not have the extra gene.

Advantages and Disadvantages:

Advantages and Disadvantages Can invade slow dividing cells e.g. lung cell, skin cell etc. Less immune rejection than retroviruses Gendicine , adenoviral p53 gene therapy for head and neck cancer in China in 2003. Multiple settings of therapy needed….

Herpes simplex virus:

Herpes simplex virus Double stranded DNA virus that attack neurons (neurotropic) Helpful for gene delivery in CNS conditions

Non viral methods of gene delivery:

Non viral methods of gene delivery Direct introduction of therapeutic cDNA into target cells. Advantage of low host immunogenicity can be used only with certain tissues and requires large amount of DNA

Physical methods:

Physical methods Injection of naked DNA - IM injection of naked DNA plasmid - Low success rate Electroporation -pulses of high voltage to carry DNA across membrane - efficient and works across wide range of cell types

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Gene gun - DNA coated with gold particles and loaded into device which generates force to achieve penetration of DNA into cells

Chemical methods:

Chemical methods Lipoplexes and polyplexes - cationic lipids due to their positive charge, used to condense negatively charged DNA so as to fascilitate encapsulation of DNA into liposomes - endocytosis of liposomes followed by lysis releases DNA into cytoplasm

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Detrimers - highly branched macromolecules with spherical shape - cationic detrimers preffered - Detrimer DNA complex taken by endocytosis, releases DNA in cytoplasm of desired cell

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The 47 th chromosome - Introduce the artificial chromosome into cell by microinjection - could segregate and replicate like other chromosomes, hence stable - can carry large amount of DNA - so far done only in mice

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Ormosil - organically modified silica or silicate - used as DNA vectors and deliver DNA loads specifically to targeted cell in living animals

Which diseases can be treated ???:

Which diseases can be treated ??? • About 4000 diseases have been traced to gene disorders • Current and possible candidates for gene therapy include cancer, AIDS, cystic fibrosis, Parkinson's, Alzheimer’s disease, sickle cell disease, thalasemia etc.

Major developments in gene therapy:

Major developments in gene therapy The first case - Performed on Sept. 14, 1990 at National institute of health US - Ashanti DeSilva, 4 yrs, treated for SCID - Doctors removed WBCs from patient, inserted missing gene into WBC and put it back into bloodstream - Boosted her immunity - Successful but short lasting !!!


Hemophilia Autosomal recessive disorder due absence of factor VIII leading to bleeding tendencies Gene therapy offers hope !!!

Cystic fibrosis:

Cystic fibrosis Autosomal recessive, mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein located on chromosome 7 Defective exocrine glands function 3 successful trials done

Severe combined immunodeficiency:

Severe combined immunodeficiency Patient prone to life threatening infections due to faulty WBC function Deficiency of adenosine deaminase Replacement of abnormal gene has solved the problem in 1993

Parkinson’s disease:

Parkinson’s disease Loss of DA neurons in substantia nigra cells resulting into classical symptom In 2000, scientists have tried inserting genes controlling DA secretion into brain using liposomes coated in polymer Significant achievement because viral vectors are too big to cross BBB

Cancer therapy:

Cancer therapy Abnormal growth of cells due to loss of cell division control Trial conducted in May 2007, successfully controlled growth of lung cancer Similar trials going on to control head and neck tumors, prostate cancer, leukemias and many more…..

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Genetic modification of tumor - introduction of tumor suppressor gene e.g. p53 - Antisense therapy, to block the translation of defective gene - Suicidal genes Anti-angiogenic gene therapy - inhibition of production of angiogenic cytokine e.g. VHL gene - introduction of genes with antiangiogenic properties e.g. thrombospondin, interferon etc.

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Genetic modification of immune system - By inserting TNF α , IL-2 and other cytokine genes into tumor cells to increase the immune recognition and destruction by lymphocytes Genetic protection of normal tissues from cytotoxic drugs - Introduction of MDR-1 gene into bone marrow cells, making them less susceptible to destruction to anticancer agents

Antisense therapy:

Antisense therapy Very promising approach to block the expression of particular defective gene by introducing antisense oligonucleotides 15-25 base pair single stranded nucleotides that interact with certain segments of specific genes and prevents their translation

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Fomivirsen is an antisense oligonucleotide approved for CMV retinitis VRX496 , gene based immunotherapy for HIV Uses lentivirus for delivery of antisense gene against HIV envelop

Suicide gene therapy:

Suicide gene therapy Gene that activates anticancer prodrug is introduced in cancer cell along with anticancer drug. Transfer of the herpes simplex thymidine kinase gene (HSVtk) into the tumor cells using retroviral or adenovirus vectors followed by the administration of ganciclovir (GCV) provides a potential strategy for the treatment of some malignancies

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GCV is metabolized by the viral thymidine kinase into active triphosphate form. GCV-triphosphate is a potent inhibitor of viral DNA-polymerase cause cell growth inhibition and cell death. Therefore, cells that express HSVtk become sensitive to the toxic effect of GCV and can be eradicated by in vivo by the administration of GCV

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However, during GCV treatment, not only the cells that express HSVtk gene are killed, but also frequently neighbouring tumor cells that are not genetically altered.”This phenomenon is called “ bystander effect”

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Currently various trials going on - Lesch Nyhan syndrome - Stroke, multiple sclerosis by delivering nerve growth factor gene - IDDM - DMD

Problems with gene therapy:

Problems with gene therapy Short lived nature - Before gene therapy can become permanent cure of any condition, therapeutic DNA introduced must remain functional and host cell should be long lived and stable - multiple rounds of therapy required

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Immune response - any foreign object elicit immune response - if rejection occurs in patient, repeat therapy also difficult

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Problems with viral vectors - Toxicity, immune and inflammatory response - once inside the patient, may recover its ability to cause disease ??

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Multigenic disorders - single gene disorders best for treatment - Unfortunately, most commonly occurring disorders like heart diseases, HTN, DM are combined effects of variation in many genes

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Chance of inducing tumor (insertional mutagenesis) - insertion of gene at wrong place could disturb function of normal gene - e.g. altered p53 function has lead to leukemia

Major setbacks !!!:

Major setbacks !!! In 2000, death of Jesse Gelsinger during gene therapy for SCID due to multiorgan failure 3 Patients suffered from leukemia (intertional mutagenesis)

Costly affair !!!:

Costly affair !!! Though still in clinical phases…. Very costly Only for high society ???


Conclusion Very promising approach Still in its infancy….. Various ‘pros and cons’, ethical issues “ Gene therapy will revolutionize medicine over next ten to twenty years, but the big question is….When ???” - French Anderson, 2001.


References Mary Relling, Kathleen Giacomini, Pharmacogenetics; Goodman and Gilmans , Pharmacological basis of therapeutics; 12;145-68;2010. Micheal Aminoff, Pharmacologic management of parkinsonism; Bertram G. Katzung , Basic and clinical pharmacology:11:478;2009

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Gene therapy, K.D.Tripathi , Essentials of medical pharmacology;520-22;6;2009. Mark Kelmann, New method of gene therapy for melanoma; The Nature ; 67;12-17;2009. Edvard Shane, Antisense therapy for HIV; NEJM ; 35;23-30;2008 Robert Johnson, Approach to gene therapy; The Scientist ; 108; 2008.



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