Gene Therapy Resala

alaa essa2008alaa essa2008


Zagazig Univeristy

Fuculty of Medicine


An essay submitted

By

Alaa Ibrahim Essa

Demonstrator of pharmacologyDemonstrator of pharmacology


Gene TherapyGene Therapy A promising A promising Approach in Approach in

Prevention and Prevention and Treatment of the Treatment of the

DiseasesDiseases


Under supervision ofUnder supervision ofUnder supervision ofUnder supervision of

Nabila Hassan El-BarodyProf. 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 Gene therapy intends to treat or alleviate disease by genetically alleviate disease by genetically modifying the cells of the patients.modifying the cells of the patients.

One of the goals of gene therapy is One of the goals of gene therapy is

to develop treatment for diseases to develop treatment for diseases for which there is for which there is no classical no classical treatment available. treatment available.


GeneGene therapy has distinct potential to therapy has distinct potential to treat different types of treat different types of malignancies malignancies

Gene therapy offers the promise of Gene therapy offers the promise of an effective cure for both an effective cure for both genetic and genetic and acquired brain diseases acquired brain diseases

Some preclinical & clinical studies Some preclinical & clinical studies suggest that genesuggest that gene therapy may be a therapy may be a useful therapeutic approach to useful therapeutic approach to ischemic heart diseaseischemic heart disease


Aim of the workAim of the work

The aim of this work is to expresses The aim of this work is to expresses the technology behind gene therapy the technology behind gene therapy and provides examples of how the and provides examples of how the approach is being used in clinical approach is being used in clinical medicine for prevention & treatment medicine for prevention & treatment a variety of genetic diseases a variety of genetic diseases

also we try to determine the different also we try to determine the different methods of gene delivery methods of gene delivery


History of Gene TherapyHistory of Gene Therapy


On September 1990On September 1990, the first approved gene , the first approved gene therapy clinical trial took place when therapy clinical trial took place when Ashanthi DeSilvaAshanthi DeSilva, a 4-year-old girl with , a 4-year-old girl with Adenosine DeaminaseAdenosine Deaminase deficient which deficient which causes causes Severe Combined Immunodeficiency Severe Combined Immunodeficiency diseasedisease treated by her own T cells treated by her own T cells engineered with a retroviral vector carrying engineered with a retroviral vector carrying a normal ADA genea normal ADA gene


Jesse GelsingerJesse Gelsinger (June 18, 1981 – (June 18, 1981 – September17 , 1999) was the first person September17 , 1999) was the first person publicly identified as having died in a publicly identified as having died in a clinical trial for gene therapy.clinical trial for gene therapy.

He died after having suffered a He died after having suffered a massive massive immune responseimmune response triggered by the use of triggered by the use of the viral vector used to transport the gene the viral vector used to transport the gene into his cells. into his cells.


Introduction to Introduction to Medical GeneticsMedical Genetics


Base pairingBase pairing

Each type of base on one strand Each type of base on one strand forms a bond with just one type of forms a bond with just one type of base on the other strand. This is base on the other strand. This is called complementary base pairing. called complementary base pairing. where where AA bonding only to bonding only to TT, and , and CC bonding only to bonding only to GG. .


GenomesGenomes

The genetic information in a genome is The genetic information in a genome is held within held within genesgenes, and the complete , and the complete set of this information in an organism set of this information in an organism is called its is called its genotypegenotype. A gene is a . A gene is a unit of unit of heredity heredity and is a region of and is a region of DNA that encodes the production of DNA that encodes the production of specific specific proteinprotein in an organism. in an organism.


DNA-modifying enzymesDNA-modifying enzymes

1- Nucleases and ligases 1- Nucleases and ligases 2- Topoisomerases2- Topoisomerases3- Polymerases3- Polymerases a- a- DNA-dependent DNA DNA-dependent DNA

polymerasepolymerase b- RNA-dependent DNA b- RNA-dependent DNA

polymerasespolymerases c- DNA-dependent RNA c- DNA-dependent RNA

polymerase polymerase


RNA versus DNARNA versus DNA

1- Unlike 1- Unlike DNADNA, which is , which is double-strandeddouble-stranded, , RNARNA is a is a single-stranded single-stranded molecule molecule

2- While 2- While DNADNA contains contains deoxyribosedeoxyribose, , RNARNA contains contains riboseribose

3- The complementary nucleotide to 3- The complementary nucleotide to adenineadenine is not is not thyminethymine but rather but rather uraciluracil


Gene Delivery Gene Delivery System System


An ideal gene delivery method has An ideal gene delivery method has three major criteria:three major criteria:

1- 1- protectprotect the transgene against the transgene against degradation by degradation by nucleasesnucleases in intercellular in intercellular matrices.matrices.

2- 2- bringbring the the transgenetransgene across the plasma across the plasma membrane and into the nucleus of target membrane and into the nucleus of target cells.cells.

3- It should have 3- It should have no serious adverse no serious adverse effectseffects..


Delivery methodsDelivery methods


Gene therapyGene therapy

In vivo Ex vivo


There are some important characters should be There are some important characters should be present in virus to be present in virus to be identical vectoridentical vector in gene in gene therapy:-therapy:-

1- safety1- safety:-:-

by deleting the viral genome by deleting the viral genome critical for viral replicationcritical for viral replication

2- low toxicity2- low toxicity..

3- stability3- stability..

4- cell type specificity (4- cell type specificity (tissue tropismtissue tropism).).


Some of viruses used in Some of viruses used in gene therapy:-gene therapy:-

1- 1- Retroviruses:Retroviruses: Retroviruses have a single stranded RNA Retroviruses have a single stranded RNA

genomegenome


Advantages of retrovirus gene Advantages of retrovirus gene therapy:therapy:

Good at Good at targetingtargeting and entering cells and entering cells Can Can target specific cellstarget specific cells through modification of through modification of

surface proteins surface proteins Can be modified to Can be modified to not replicatenot replicate within host cells within host cells

Disadvantages:Disadvantages: Can trigger Can trigger an immune responsean immune response within the host within the host Can't be used to carry Can't be used to carry larger geneslarger genes No No longlong-term benefits. -term benefits. The The random insertionrandom insertion of genes can disrupt other of genes can disrupt other

genes. genes.


AdenovirusesAdenoviruses Vectors: Vectors: Adenovirus is Adenovirus is double-stranded DNAdouble-stranded DNA virus. virus.

GutlessGutless vectors have all viral DNA ( vectors have all viral DNA (vDNAvDNA) ) eliminated to improve eliminated to improve safetysafety and and immunogenicityimmunogenicity. .


Advantages of Using Adenoviruses:Advantages of Using Adenoviruses:

1-1- Easy production Easy production of virus in high titres of virus in high titres

2-2- high transfection efficiency high transfection efficiency

3- Ability to be expressed in both 3- Ability to be expressed in both proliferating and proliferating and non-proliferatingnon-proliferating cells cells

Disadvantages and Problems:Disadvantages and Problems:

1- Highly 1- Highly antigenicantigenic..

2-2-InflammatoryInflammatory response elicited by their injection response elicited by their injection

3- 3- Immune responseImmune response developed due to the developed due to the inflammationinflammation

4-4- Reduced expressionReduced expression so need frequent doses. so need frequent doses.


Nonviral delivery Nonviral delivery methodsmethods:-:-

Gene Transfer by Needle Injection Gene Transfer by Needle Injection of Naked DNA:- of Naked DNA:-

attractive because of its attractive because of its simplicitysimplicity and and lack of lack of toxicitytoxicity..

airwayairway gene delivery and gene delivery and intramuscular intramuscular injection of naked DNA for the treatment of injection of naked DNA for the treatment of acute diseasesacute diseases and and DNA-based DNA-based immunizationimmunization depends upon direct injection depends upon direct injection of naked DNA. of naked DNA.


Gene Transfer by Physical Gene Transfer by Physical Methods:-Methods:-

1-Transfer by Gene Gun1-Transfer by Gene Gun


2-Gene Transfer by 2-Gene Transfer by Electroporation:-Electroporation:-

increase in the electrical conductivityincrease in the electrical conductivity by an by an externally applied electrical field.externally applied electrical field.


The advantages of using of The advantages of using of electroporation:-electroporation:-

1-DNA as 1-DNA as largelarge as 100 kb has been effectively as 100 kb has been effectively delivered into muscle cells. delivered into muscle cells.

2-Long-term expression2-Long-term expression over 1 year over 1 year

Drawbacks for in vivo Drawbacks for in vivo application of electroporationapplication of electroporation:-:-

1- 1- limitedlimited effective effective rangerange of 1 cm between the of 1 cm between the electrodes electrodes

2-2-surgical surgical procedure is required to place the procedure is required to place the electrodes deep into the internal organs. electrodes deep into the internal organs.


3-Ultrasound-Facilitated Gene 3-Ultrasound-Facilitated Gene TransferTransfer

ultrasoundultrasound creates creates membrane poresmembrane pores and and facilitates intracellular gene transfer through facilitates intracellular gene transfer through passive diffusionpassive diffusion of DNA across the of DNA across the membrane pores membrane pores

It could become an ideal method for It could become an ideal method for noninvasivenoninvasive gene transfer into cells of the gene transfer into cells of the internal organsinternal organs. .


4-Hydrodynamic Gene Delivery4-Hydrodynamic Gene Delivery

simple method that introduces naked plasmid simple method that introduces naked plasmid DNA into cells in DNA into cells in highly perfusedhighly perfused internal internal organs (e.g., organs (e.g., the liverthe liver) )

delivery efficiency is determined by the delivery efficiency is determined by the anatomic structureanatomic structure of the organ, the of the organ, the injection volumeinjection volume, and the , and the speed of injectionspeed of injection

The gene transfer efficiency of this simple The gene transfer efficiency of this simple procedure is the procedure is the highesthighest among the nonviral among the nonviral methods. methods.


Gene Delivery by Chemical Gene Delivery by Chemical Methods:-Methods:-

1- Lipoplexes:-1- Lipoplexes:-Plasmid DNA covered with Plasmid DNA covered with lipidslipids

There are three types of lipids could be used:-There are three types of lipids could be used:-

a- Anionic b- Neutral c- Cationic.a- Anionic b- Neutral c- Cationic.

2-2- Polyplexes Polyplexes:-:- Complexes of Complexes of polymerspolymers with DNA with DNA

Most polyplexes consist of Most polyplexes consist of cationic polymers.cationic polymers.


Cancer gene Cancer gene therapytherapy


Cancer Gene therapy Cancer Gene therapy strategies:-strategies:-

fourfour separate pathways have been developed, separate pathways have been developed, namely :-namely :-

1- 1- immunoimmuno-therapy -therapy

2- introduction of tumor suppressor genes\ 2- introduction of tumor suppressor genes\ induction of induction of apoptosisapoptosis

3- enzyme 3- enzyme prodrugprodrug therapy therapy

4- inhibition of tumors 4- inhibition of tumors angiogenesisangiogenesis..


Immunotherapy

The induction of cytokine

Genetic modification of lymphocytes

Tumor antigen vaccines


A) Induction of cytokine or co-A) Induction of cytokine or co-stimulatory molecule expressionstimulatory molecule expression-:-:

The frequently employed cytokines include The frequently employed cytokines include interleukin2 interleukin2 ( IL-2( IL-2)),, interleukin 12 interleukin 12 ( IL-12( IL-12)) & & granulocyte-macrophage colony-stimulating granulocyte-macrophage colony-stimulating factor factor (GM-CSF(GM-CSF))..

Many clinical protocols involve an Many clinical protocols involve an ex vivo ex vivo approachapproach


B) Genetic modification of lymphocytes:-B) Genetic modification of lymphocytes:-

Currently extremely popular is the modification Currently extremely popular is the modification of of denderitic cells (DCs)denderitic cells (DCs)

DCsDCs cells are bone marrow-derived cells cells are bone marrow-derived cells function as extremely potent function as extremely potent APCsAPCs and and capable of activating capable of activating T-cells. T-cells.

Intratumoural injection of DCs modified with an Intratumoural injection of DCs modified with an adenovirus encoding adenovirus encoding CD40 ligandCD40 ligand led to led to significant tumor regression in a murine model significant tumor regression in a murine model


C) Tumour antigen vaccines:-C) Tumour antigen vaccines:-

both the both the DCDC work work and the re-introduction of and the re-introduction of GM-CSFGM-CSF transduced tumour cells could be transduced tumour cells could be classified as vaccination strategiesclassified as vaccination strategies

synthetic peptidessynthetic peptides are the most effective are the most effective strategy for immunization aginst cancerstrategy for immunization aginst cancer

Peptides can be coated onto Peptides can be coated onto dendritic cellsdendritic cells to to bypass any defect in antigen presenting cell bypass any defect in antigen presenting cell function related to the presence of the cancer function related to the presence of the cancer


II- Introduction of tumor suppressor II- Introduction of tumor suppressor genes & induction of apoptosis:-genes & induction of apoptosis:-

MutationsMutations in in p53p53 were found in were found in 40% to 60%40% to 60% of of patients with patients with colorectalcolorectal cancer. cancer.

Generally, mutation or over expression of p53 Generally, mutation or over expression of p53 seems to be associated with an seems to be associated with an unfavourableunfavourable prognosis for patients with cancer colon.prognosis for patients with cancer colon.

However, there are also investigations with However, there are also investigations with contrarycontrary results or results or withoutwithout any associations. any associations.


III- Virus-directed enzyme\prodrug III- Virus-directed enzyme\prodrug therapy:-therapy:-

The principle is to achieve tumor cell–selective The principle is to achieve tumor cell–selective activation of prodrugs and to produce activation of prodrugs and to produce tumor-specific tumor-specific cytotoxicity cytotoxicity

Potentially, one of several Potentially, one of several ""vectorsvectors" can be used to " can be used to deliver a deliver a prodrug-activating enzymeprodrug-activating enzyme selectively to selectively to tumor foci tumor foci in vivoin vivo

the elevated level of enzyme at the tumor foci the elevated level of enzyme at the tumor foci produce produce high local concentrationshigh local concentrations of active drug, of active drug, increasing the antitumor effect and decreasing the increasing the antitumor effect and decreasing the toxicity of the systemically given prodrug. toxicity of the systemically given prodrug.



Gene therapy in the Gene therapy in the nervous systemnervous system


The predominant The predominant challengechallenge in this field is to find in this field is to find effective effective vectorsvectors. The reasons for this are : . The reasons for this are :

1- Neurons in the adult brain are 1- Neurons in the adult brain are postmitoticpostmitotic..

2- the brain is far 2- the brain is far less accessibleless accessible than other than other organs.organs.

3- The brain is immensely 3- The brain is immensely heterogeneousheterogeneous..

4- Because of their size, elongated morphology, 4- Because of their size, elongated morphology, and high metabolic demands, neurons are and high metabolic demands, neurons are fragile cellsfragile cells. .


Gene therapy strategies in Gene therapy strategies in neurological insult:-neurological insult:-

Most gene therapy studies in the nervous Most gene therapy studies in the nervous system have made use of one of system have made use of one of threethree approaches approaches


Step 1Step 1: : FibroblastsFibroblasts are taken from the individual. are taken from the individual. Step 2Step 2: They are engineered to express the : They are engineered to express the

protectiveprotective transgenetransgene.. Step 3Step 3: The altered fibroblasts are : The altered fibroblasts are transplantedtransplanted

into the relevant into the relevant brain regionbrain region. .

A) The first approachA) The first approach


B) A second approachB) A second approach

Step 1Step 1: A protective transgene is encased in a : A protective transgene is encased in a liposomal shell.liposomal shell.

Step 2Step 2: It is introduced into the : It is introduced into the nervous systemnervous system.. Step 3Step 3: The liposome shell merges with the plasma : The liposome shell merges with the plasma

membrane of the target neuron, releasing the membrane of the target neuron, releasing the transgenetransgene into the cell. into the cell.


C) The third approachC) The third approach

Step 1Step 1: A protective transgene is incorporated into the : A protective transgene is incorporated into the genome of a virus genome of a virus

Step 2Step 2:: The recombinant DNA is then encased in a viral The recombinant DNA is then encased in a viral coat, forming a coat, forming a viral vectorviral vector. .

Step 3Step 3: The vector is introduced into the : The vector is introduced into the nervous systemnervous system, , where it is then endocytosed into target cells where it is then endocytosed into target cells

Step 4Step 4: : DNADNA is released into the target cell. is released into the target cell. Step 5Step 5:: DNA is translocated to the DNA is translocated to the nucleusnucleus. .


Gene therapy for Parkinson's Gene therapy for Parkinson's disease: -disease: -

TowTow gene therapy strategies have been gene therapy strategies have been investigated :-investigated :-

1-1-Tyrosine hydroxylaseTyrosine hydroxylase ( TH) ( TH) transfer to the transfer to the nigrostriatal pathway either by virus or nigrostriatal pathway either by virus or liposomes.liposomes.

22- Introduction of - Introduction of neuroprotective genesneuroprotective genes: : Glial Glial cell line – derived neurotrophic factor cell line – derived neurotrophic factor (GDNF)(GDNF) is mainly used. is mainly used.


Gene therapy for AlzheimerGene therapy for Alzheimer::

Neurotrophic geneNeurotrophic gene therapy strategies using therapy strategies using nerve growth factor nerve growth factor ((NGFNGF)) and brain-derived and brain-derived neurotrophic factor neurotrophic factor (BDNF)(BDNF) are the main used are the main used strategies with gene delivery directly into the strategies with gene delivery directly into the medial septum via recombinant adeno- medial septum via recombinant adeno- associated viral vector. associated viral vector.

These measures resulted in attenuation of These measures resulted in attenuation of cholinergic cellcholinergic cell loss loss


Gene therapy in epilepsy:-Gene therapy in epilepsy:-

Mainly in Mainly in temporal lobe epilepsytemporal lobe epilepsy. By . By introduction of Glial cell line–derived introduction of Glial cell line–derived neurotrophic factor neurotrophic factor (GDNF)(GDNF) into the into the rat rat hippocampushippocampus suppressing the seizures of suppressing the seizures of temporal lobe epilepsy models either before temporal lobe epilepsy models either before hippocampal kindlinghippocampal kindling or after kindling. or after kindling.


Gene therapy in strokeGene therapy in stroke:-:-

Gene therapy strategies in treating stroke depend Gene therapy strategies in treating stroke depend upon the delivery of upon the delivery of neuroprotective genesneuroprotective genes at at high levels high levels

These genes may causeThese genes may cause:-:-

a- a- energy restorationenergy restoration by the glucose by the glucose transporter transporter (GLUT-1)(GLUT-1)

b- buffering calcium excess by b- buffering calcium excess by calbindincalbindin..

c- inhibiting apoptotic death by c- inhibiting apoptotic death by BCL-2BCL-2


Gene therapy in Gene therapy in cardiovascular cardiovascular

systemsystem


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Gene Therapy Resala

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