What is the Gene Therapy?

It is a technique for correcting defective genes that are responsible for disease development, it mean using genes to treat or prevent disease

• There are three approaches:

1. Replacing a mutated gene that causes disease with a healthy

copy of the gene.

2. Inactivating, or “knocking out,” a mutated gene that is

functioning improperly.

3. Introducing a new gene into the body to help fight a disease.

In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery,

Gene Therapy

1. Scientists focused on diseases caused by single-gene defects, such as* cystic fibrosis* haemophilia* thalassemia* sickle cell anemia

2. Treatment other diseases

• inherited disorders

• some types of cancer

• viral infections

Which diseases can be treated?

• The first gene therapy was performed on

September 14th, 1990

– Ashanti DeSilva was treated for SCID

• Sever combined immunodeficiency

– Doctors removed her white blood cells,

inserted the missing gene into the WBC, and

then put them back into her blood stream.

– This strengthened her immune system

– Only worked for a few months

The First Case

• Gene transplantation

(to patient with gene deletion)

• Gene correction

(To revert specific mutation in the gene of interest)

• Gene augmentation

(to enhance expression of gene of interest)

Gene therapy could be very different for different diseases

the therapeutic genes are transferred into the somatic cells of a

patient. Any modifications will be restricted to the individual patient

only, and will not be inherited to offspring.

Several experiments are currently in clinical

trials with varied success. Over 600 clinical trials

utilizing somatic cell therapy are underway in the United States

Somatic gene therapy

Germ cells (sperm or eggs) are modified by the introduction of functional

genes, which are integrated into their genomes. if a germ cell is genetically

modified then all the cells in the organism will contain the modified gene.

This would allow the therapy to be heritable and passed on to later

generations. Although this should be highly effective in counteracting

genetic disorders and hereditary diseases, some country prohibit this

application in human beings, at least for the present, for technical and

ethical reasons, including insufficient knowledge about possible risks to

future generations and higher risk than somatic gene therapy,

Germline gene therapy

Types of gene therapy

In these process bacterial DNA is moved from one

bacterium to another by a virus (a bacteriophage).

Methods of gene therapy

Methods of gene therapy

This process is relatively common in bacteria, but less so in eukaryotes. Transformation is often used in laboratories to insert novel genes into bacteria for experiments or for industrial or medical applications.

Methods of gene therapy

Vector

All viruses bind to their hosts and introduce their genetic material into

the host cell as part of their replication cycle, First removing the viral

DNA that cause disease replace those genes with genes encoding

the desired effect and using the virus as a vehicle to deliver the

therapeutic DNA. This genetic material contains basic 'instructions' of

how to produce more copies of these viruses,the host cell will carry

out these instructions and produce additional copies of the virus,

leading to more and more cells becoming infected.

A number of viruses have been used for human gene therapy,

includingretrovirusadenoviruslentivirusherpes simplex viruspox virusadeno-associated virus.

Viral vectors

Viral vectors

P53 tumor suppressor gene

• When in mutates, p53 can lose its suppressive powers which effects of abnormal cell growth

• P53 is the most commonly mutated gene found in human tumors

Direct injection to tumor

The adenovirus containing the p53 gene binds to the receptor in the cell membrane of the cancer cell

Mechanisms

Mechanisms

The adenovirus injects its gene, which now includes the p53 tumor suppressor gene. In to the cell nucleus

P53 expressed to produce protein damage DNA and cell suicide (apoptosis)

Mechanisms

•Over 16,000 patients alteady treated•No serious side effects•Common side effects is self limited fever

P53 gene therapy is safe?

1. Viruses can usually infect more than one type of cell. when

viral vectors are used to carry genes into the body, they might infect

healthy cells as well as cancer cells

2. the new gene might be inserted in the wrong location in the DNA,

possibly causing harmful mutations to the DNA .

3. there is a slight chance that this DNA could be introduced into the

patient’s reproductive cells. If this happens, it could produce changes

that may be passed to offspring

4. transferred genes could be overexpressed, producing so much of the

missing protein as to be harmful.

5. the viral vector could cause an immune reaction;

6. and that the virus could be transmitted from the patient to other

individuals or into the environment.

Disease Defect Target cell

immunodeficiency Bone marrow cells

T-lymphocytes

Hemophilia Liver, muscle

Cystic fibrosis Lung Cells

Cancer Many cell types

Neurological diseases Parkinson’s/

Alzheimers

Nerve Cells

Infectious diseases AIDS, hepatitis B White Blood Cells

• Spontaneous uptake by endocytosis

• Plasmid liposome complex

• Uncovered plasmids

• Gene gun methods

• electroporation

Is a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing DNA to be introduced into the cell

Electroporation

Introducing Chromosome

To improve and facilitated the delivery of the new DNA into the cell, the DNA must be protected from damage. DNA can be covered with lipids like a liposome, is complex DNA with liposome is called lipoplexThe most common use of lipoplexes has been in gene transfer into cancer cells, where

activated tumor suppressor genes in the cell and decrease the activity of oncogenes

Called naked DNA or vector DNA, it is histonefree DNA that is passed from cell to cell during a gene transfer

Direct injection of free DNA into certain tissues, particularly muscle, the simplicity of this technique has led to its adoption in a number of clinical protocols, this technique has been applied to the gene therapy of cancer where the DNA can be injected directly into the tumour

DNA plasmid vector vaccines carry the genetic information encoding an antigen, allowing the antigen to be produced inside of a host cell, leading to a cell-mediated immune response via the MHC I pathway. The plasmid DNA vaccine carries the genetic code for a piece of pathogen or tumor antigen. The plasmid vector is taken up into cells and transcribed in the nucleus

DNA vaccines or plasmid vector

1. amplifying bacterial plasmid, The bacterial plasmids inserts using recombinant DNA technology, where bacterial growth produces multiple plasmid copies. The plasmid DNA is then purified from the bacteria, by separating the circular plasmid from bacterial DNA and other bacterial impurities. This purifies DNA acts as the vaccine

2.delivery of plasmids through intramuscular, intradermal and intravenous injection. The skin and mucous membranes are the best site for immunization due to the high concentrations of dendritic cells (DC), macrophages and lymphocytes . Intradermal injection of DNA with a gene gun have been used.

Mechanisms

1. The single stranded mRNA2. is translated into protein in the

cytoplasm. The DNA vaccine-derived protein antigen

3. is then degraded by proteases into intracellular peptides

4. The vaccine derived-peptide binds MHC class I molecules

5. Peptide antigen/MHC I complexes are presented on the cell surface

6. binding cytotoxic CD 8+ lymphocytes, and inducing a cell-mediated immune response., . CTLs inhibit viruses through both cytolysis or by cytokine production ( chemokines, interferons,interleukins,lymphokines, tumour necrosis factor)

Mechanisms

• The foreign protein can also be presented by the MHC class II pathway by APCs which elicit helper T cells (CD4+) responses. These CD4+ cells are able to recognize the peptides formed from exogenous proteins that were endocytosed or phagocytosed by APC, then degraded to peptide fragments and loaded onto MHC class II molecules. Depending on the the type of CD4+ cell that binds to the complex, B cells are stimulated and antibody production is stimulated. This is the same manner in which traditional vaccines work

• induce the expression of antigens that resemble native viral epitopes more closely than standard vaccines do since live weakened or killed vaccines are often altered in their protein structure and antigenicity.

• Plasmid vectors can be constructed and produced quickly and the coding sequence can be manipulated in many ways, costs lower than traditional vaccines.

• DNA vaccines encoding several antigens or proteins can be delivered to the host in a single dose.

• they are also very temperature stable making storage and transport much easier.

• The continues expression of the viral antigen caused by gene vaccination in an environment containing many APCs (antigen processing cell) may promote successful therapeutic immune response which cannot be obtained by other traditional vaccines .

Advantages

Although DNA can be used to raise immune responses against pathogenic proteins, certain microbes have outer capsids that are made up of polysaccharides. This limits the extent of the usage of DNA vaccines because they cannot substitute for polysaccharide-based subunit vaccines .

Limitations

• Short Lived – Hard to rapidly integrate therapeutic DNA into genome and rapidly

dividing nature of cells prevent gene therapy from long time– Would have to have multiple rounds of therapy

• Immune Response– new things introduced leads to immune response– increased response when a repeat offender enters

• Viral Vectors– patient could have toxic, immune, inflammatory response– also may cause disease once inside

• Multigene Disorders– Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes

are hard to treat because you need to introduce more than one gene• induce a tumor if integrated in a tumor suppressor gene because

insertional mutagenesis

Problems with Gene Therapy

Lectins in gene therapy

Lectins are carbohydrate-binding proteins or glyco-

proteins binding selectively without the involvement of

enzymes specific for carbohydrate ligands. Lectins are

present in organisms and play very important roles in life,

Lectins' functioning mechanism is based on the reaction of

glycolysation

Gene responsible for expression

lection found in chromosome 10q11.2-

q21

Found in plnats grains, legume, soy

bean, kidney bean

• Recognition of cells, carbohydrates, and proteins

• Bind soluble extracellular and intercellular glycoproteins.

• The regulation of cell adhesion

• Cell differentiation

• lectins are found on the surface of liver cells that specifically recognize galactose residues. It is believed that these cell-surface receptors are responsible for the removal of certain glycoproteins from the circulatory system.

• Control of protein levels in the blood.

Lectins recognize tumor marker which play important role for diagnosing tumor cell, screening tumour and able to detect subtle neoplastic changes

Tumour marker are molecules that can be detected in blood fluids or tissue, which are produced as a response to cancer or by caner cells themselves

mannose-bindig lectins are interacting with

the carbohydrate chains Lectin has

glycoconjugates property which bind to

different carbohydrate, lectins bind to cell

membrane carbohydrate (antigen), tumor cell

has altered antigen with the use of lectin affinity

chromatography (LAC) and histochemistry

methods by comparing tumor antigen with

normal cell antigen, lectins provide practical

application during observation of changes

occurring in the cell membrane in different

stages of physiological and pathological

development of human cell, and tumour

diagnosis and scanning

Lectin suppress cancer cell growth and

metastasis by blocking polyamines

• Polyamines growth factor are synthesized in the body or

derived form the diet, or produce by bacteria in the gut, or

by cancer cell they are essential to cellular proliferation and

differentiation

• Lectin cause (polyamine deprivation) by decreasing

digestion of polyamines then cell growth is stopped

• Cancer cell produce polyamines, which adsorbed by lymphocyte lead to

1. inhibit anti-tumor cells called natural Killer cells

2. metastasis (tumor cell spread) when polyamines level increase immune cells fail to recognize cancer cell antigen and decreased cytotoxic activity of killer cell

Mannose-binding lectin (MBL), is a lectin that is induce innate immunity, a low expression of an MBL1 was detected in liver.

• mutations in exon 1 of the human MBL2 gene at codon 52 reduce the level of functional serum MBL by disrupting the collagenous structure of the protein.

• nucleotide substitutions in the promoter region of the MBL2 gene affect the MBL serum concentration

MBL belongs to the class of collectins in the C-type lectin, whose function is carbohydrate antigen recognition of bacteria, viruses, and fungi. MBL in the first line of defense results in activation of the lectin pathway of the complement system.

The polymorphisms of exon 1 cause susceptibility to various common infections, including meningococcal disease.

lectins inducing apoptosis , lectin found in the seeds of the castor plant. Has cytotoxic effect that kill human cell , a lethal dose of purified ricin is approximately 1 milligram per kilogram of body weight.

• Lectin toxicity is by inhibiting protein synthesis

1. with immuno-modulatory or antiproliferative activity lectin belonging to II-RIP class (Ribosome Inactivating Proteins)

2. prevent assembling amino acids to proteins during translation of mRNA or by cause cell death (a ppotosis) this cell-killing activity of lectin make it useful for treatment of tumor cell.

1. Cancer treatment by castor Lectins

Lectins found in Mistletoe plant induce apoptosis by

1. inhibition of telomerase activity

2. promote of p53- and p21 independent pathway (suppresser gene)

Telomerase Is the enzyme that add repeats DNA sequence to the 3' end of DNA strands that promote chromosomal stability, Telomere shortness in human is a marker of disease and cancer.

2. Cancer treatment by Mistletoe Lectins

Plant Lectin PHA (phytohaemagglutinin) found in legume has carbohydrate-binding specificity for a complex oligosaccharide containing galactose, and mannose, possess anticancer properties in vitro, in vivo, bind to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis, and inhibition of tumor growth.

• Has mitogenic effect in lymphocyte which trigger lymphocyte differentiate and produce T cell and which increase immune response to tumour

3. Cancer treatment by legume Lectins

1. Lectins mediate attachment and binding of bacteria and viruses

2. Plant Lectin PHA (phytohaemagglutinin) has mitogenic effect in lymphocyte which trigger lymphocyte differentiate and produce T cell.

3. lectin is a receptor that recognizes hydrolytic enzymes containing mannose-6-phosphate, and targets these proteins for delivery to the lysosomes.

4. Lectins play important roles in the immune system by recognizing carbohydrates that are found on pathogens, Examples are the lectin complement activation pathway and mannose-binding lectin.

5. A lectin (BanLec) from bananas inhibits HIV-1 in vitro

6. They also affect the immune system by altering the production of interleukins, or activating protein kinases

• Lectins may be disabled by specific charbohydrate which bind to ingested lectins from grains, legume plants and dairy; binding can prevent their attachment to the carbohydrates within the cell membrane. Cause intestinal irritation

• Some lectins may be powerful toxins , have been incorporated into genetically engineered crops to transfer traits, such as resistance to pests and resistance to herbicides.