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Gene Therapy Resala

Gene Therapy Resala






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    Gene Therapy Resala Gene Therapy Resala Presentation Transcript

    • قالوا سبحانك لا علم لنا إلا ما علمتنا إنك أنت العليم الحكيم قالوا سبحانك لا علم لنا إلا ما علمتنا إنك أنت العليم الحكيم
    • Alaa Ibrahim Essa Demonstrator of pharmacology
    • Gene Therapy A promising Approach in Prevention and Treatment of the Diseases
    • Under supervision of Nabila Hassan El-Barody Prof. Dr. Ass. Prof Ibrahim Aly Awwad Ass. Prof Ahmed Abd El Azim Mobasher .Dr. .Dr.
    • Prof. Dr . Naamat Ahmed zakaria Prof. Dr. Moneer Nageeb Ibrahim Ass. Prof Ibrahim Aly Awwad .Dr. .Dr.
      • Gene therapy intends to treat or alleviate disease by genetically modifying the cells of the patients.
      • One of the goals of gene therapy is to develop treatment for diseases for which there is no classical treatment available.
      • Gene therapy has distinct potential to treat different types of malignancies
      • Gene therapy offers the promise of an effective cure for both genetic and acquired brain diseases
      • Some preclinical & clinical studies suggest that gene therapy may be a useful therapeutic approach to ischemic heart disease
    • Aim of the work
      • The aim of this work is to expresses the technology behind gene therapy and provides examples of how the approach is being used in clinical medicine for prevention & treatment a variety of genetic diseases
      • also we try to determine the different methods of gene delivery
    • History of Gene Therapy
      • On September 1990 , the first approved gene therapy clinical trial took place when Ashanthi DeSilva , a 4-year-old girl with Adenosine Deaminase deficient which causes Severe Combined Immunodeficiency disease treated by her own T cells engineered with a retroviral vector carrying a normal ADA gene
      • Jesse Gelsinger (June 18, 1981 – September17 , 1999) was the first person publicly identified as having died in a clinical trial for gene therapy.
      • He died after having suffered a massive immune response triggered by the use of the viral vector used to transport the gene into his cells.
      • Introduction to Medical Genetics
      • Base pairing
      • Each type of base on one strand forms a bond with just one type of base on the other strand. This is called complementary base pairing. where A bonding only to T , and C bonding only to G .
    • Genomes
      • The genetic information in a genome is held within genes , and the complete set of this information in an organism is called its genotype . A gene is a unit of heredity and is a region of DNA that encodes the production of specific protein in an organism.
    • DNA-modifying enzymes
      • 1- Nucleases and ligases
      • 2- Topoisomerases
      • 3- Polymerases
      • a- DNA-dependent DNA polymerase
      • b- RNA-dependent DNA polymerases
      • c- DNA-dependent RNA polymerase
    • RNA versus DNA
      • 1- Unlike DNA , which is double-stranded , RNA is a single-stranded molecule
      • 2- While DNA contains deoxyribose , RNA contains ribose
      • 3- The complementary nucleotide to adenine is not thymine but rather uracil
      • Gene Delivery System
    • An ideal gene delivery method has three major criteria:
      • 1- protect the transgene against degradation by nucleases in intercellular matrices.
      • 2- bring the transgene across the plasma membrane and into the nucleus of target cells.
      • 3- It should have no serious adverse effects .
      • Delivery methods
    • Gene therapy In vivo Ex vivo
      • There are some important characters should be present in virus to be identical vector in gene therapy:-
      • 1- safety :-
      • by deleting the viral genome critical for viral replication
      • 2- low toxicity .
      • 3- stability .
      • 4- cell type specificity ( tissue tropism ).
    • Some of viruses used in gene therapy:-
      • 1- Retroviruses:
      • Retroviruses have a single stranded RNA genome
      • Advantages of retrovirus gene therapy:
      • Good at targeting and entering cells
      • Can target specific cells through modification of surface proteins
      • Can be modified to not replicate within host cells
      • Disadvantages:
      • Can trigger an immune response within the host
      • Can't be used to carry larger genes
      • No long -term benefits.
      • The random insertion of genes can disrupt other genes.
    • Adenoviruses Vectors:
      • Adenovirus is double-stranded DNA virus.
      • Gutless vectors have all viral DNA ( vDNA ) eliminated to improve safety and immunogenicity .
      • Advantages of Using Adenoviruses:
      • 1- Easy production of virus in high titres
      • 2- high transfection efficiency
      • 3- Ability to be expressed in both proliferating and non-proliferating cells
      • Disadvantages and Problems:
      • 1- Highly antigenic .
      • 2- Inflammatory response elicited by their injection
      • 3- Immune response developed due to the inflammation
      • 4- Reduced expression so need frequent doses.
    • Nonviral delivery methods :-
      • Gene Transfer by Needle Injection of Naked DNA:-
      • attractive because of its simplicity and lack of toxicity .
      • airway gene delivery and intramuscular injection of naked DNA for the treatment of acute diseases and DNA-based immunization depends upon direct injection of naked DNA.
    • Gene Transfer by Physical Methods:-
      • 1-Transfer by Gene Gun
    • 2-Gene Transfer by Electroporation:-
      • increase in the electrical conductivity by an externally applied electrical field.
    • The advantages of using of electroporation:-
      • 1-DNA as large as 100 kb has been effectively delivered into muscle cells.
      • 2-Long-term expression over 1 year
      • Drawbacks for in vivo application of electroporation :-
      • 1- limited effective range of 1 cm between the electrodes
      • 2- surgical procedure is required to place the electrodes deep into the internal organs.
    • 3-Ultrasound-Facilitated Gene Transfer
      • ultrasound creates membrane pores and facilitates intracellular gene transfer through passive diffusion of DNA across the membrane pores
      • It could become an ideal method for noninvasive gene transfer into cells of the internal organs .
    • 4-Hydrodynamic Gene Delivery
      • simple method that introduces naked plasmid DNA into cells in highly perfused internal organs (e.g., the liver )
      • delivery efficiency is determined by the anatomic structure of the organ, the injection volume , and the speed of injection
      • The gene transfer efficiency of this simple procedure is the highest among the nonviral methods.
    • Gene Delivery by Chemical Methods:-
      • 1- Lipoplexes:-
      • Plasmid DNA covered with lipids
      • There are three types of lipids could be used:-
      • a- Anionic b- Neutral c- Cationic.
      • 2- Polyplexes :-
      • Complexes of polymers with DNA
      • Most polyplexes consist of cationic polymers.
    • Cancer gene therapy
    • Cancer Gene therapy strategies:-
      • four separate pathways have been developed, namely :-
      • 1- immuno -therapy
      • 2- introduction of tumor suppressor genes induction of apoptosis
      • 3- enzyme prodrug therapy
      • 4- inhibition of tumors angiogenesis .
    • Immunotherapy The induction of cytokine Genetic modification of lymphocytes Tumor antigen vaccines
    • A) Induction of cytokine or co-stimulatory molecule expression :-
      • The frequently employed cytokines include interleukin2 ( IL-2 ) , interleukin 12 ( IL-12 ) & granulocyte-macrophage colony-stimulating factor (GM-CSF ) .
      • Many clinical protocols involve an ex vivo approach
    • B) Genetic modification of lymphocytes:-
      • Currently extremely popular is the modification of denderitic cells (DCs)
      • DCs cells are bone marrow-derived cells function as extremely potent APCs and capable of activating T-cells.
      • Intratumoural injection of DCs modified with an adenovirus encoding CD40 ligand led to significant tumor regression in a murine model
    • C) Tumour antigen vaccines:-
      • both the DC work and the re-introduction of GM-CSF transduced tumour cells could be classified as vaccination strategies
      • synthetic peptides are the most effective strategy for immunization aginst cancer
      • Peptides can be coated onto dendritic cells to bypass any defect in antigen presenting cell function related to the presence of the cancer
    • II- Introduction of tumor suppressor genes & induction of apoptosis:-
      • Mutations in p53 were found in 40% to 60% of patients with colorectal cancer.
      • Generally, mutation or over expression of p53 seems to be associated with an unfavourable prognosis for patients with cancer colon.
      • However, there are also investigations with contrary results or without any associations.
    • III- Virus-directed enzymeprodrug therapy:-
      • The principle is to achieve tumor cell–selective activation of prodrugs and to produce tumor-specific cytotoxicity
      • Potentially, one of several " vectors " can be used to deliver a prodrug-activating enzyme selectively to tumor foci in vivo
      • the elevated level of enzyme at the tumor foci produce high local concentrations of active drug, increasing the antitumor effect and decreasing the toxicity of the systemically given prodrug.
      • Gene therapy in the nervous system
      • The predominant challenge in this field is to find effective vectors . The reasons for this are :
      • 1- Neurons in the adult brain are postmitotic .
      • 2- the brain is far less accessible than other organs.
      • 3- The brain is immensely heterogeneous .
      • 4- Because of their size, elongated morphology, and high metabolic demands, neurons are fragile cells .
    • Gene therapy strategies in neurological insult:-
      • Most gene therapy studies in the nervous system have made use of one of three approaches
      • Step 1 : Fibroblasts are taken from the individual.
      • Step 2 : They are engineered to express the protective transgene .
      • Step 3 : The altered fibroblasts are transplanted into the relevant brain region .
      A) The first approach
    • B) A second approach
      • Step 1 : A protective transgene is encased in a liposomal shell.
      • Step 2 : It is introduced into the nervous system .
      • Step 3 : The liposome shell merges with the plasma membrane of the target neuron, releasing the transgene into the cell.
    • C) The third approach
      • Step 1 : A protective transgene is incorporated into the genome of a virus
      • Step 2 : The recombinant DNA is then encased in a viral coat, forming a viral vector .
      • Step 3 : The vector is introduced into the nervous system , where it is then endocytosed into target cells
      • Step 4 : DNA is released into the target cell.
      • Step 5 : DNA is translocated to the nucleus .
    • Gene therapy for Parkinson's disease: -
      • Tow gene therapy strategies have been investigated :-
      • 1- Tyrosine hydroxylase ( TH) transfer to the nigrostriatal pathway either by virus or liposomes.
      • 2 - Introduction of neuroprotective genes : Glial cell line – derived neurotrophic factor (GDNF) is mainly used.
    • Gene therapy for Alzheimer:
      • Neurotrophic gene therapy strategies using nerve growth factor ( NGF ) and brain-derived neurotrophic factor (BDNF) are the main used strategies with gene delivery directly into the medial septum via recombinant adeno- associated viral vector.
      • These measures resulted in attenuation of cholinergic cell loss
    • Gene therapy in epilepsy:-
      • Mainly in temporal lobe epilepsy . By introduction of Glial cell line–derived neurotrophic factor (GDNF) into the rat hippocampus suppressing the seizures of temporal lobe epilepsy models either before hippocampal kindling or after kindling.
    • Gene therapy in stroke :-
      • Gene therapy strategies in treating stroke depend upon the delivery of neuroprotective genes at high levels
      • These genes may cause :-
      • a- energy restoration by the glucose transporter (GLUT-1)
      • b- buffering calcium excess by calbindin .
      • c- inhibiting apoptotic death by BCL-2
      • Gene therapy in cardiovascular system