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
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 .
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.
1- Nucleases and ligases
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 .
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:-
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
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.
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:-
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 :-
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)