Bio- Technology Project Work

Submitted By:
Arpita Mishra Class:- Xi th A Roll- 50

Subject Teacher: Miss Nabaneeta Das Pgt Bio-Technology

GENE THERAPY

A Chromosome

Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development. There are four approaches to gene therapy: 1. A normal gene inserted to compensate for a nonfunctional gene. 2. An abnormal gene traded for a normal gene. 3. An abnormal gene repaired through selective reverse mutation. 4. Change the regulation of gene pairs. Although the technology is still in its infancy, it has been used with some success.

 In the 1980s, Scientists began to look into gene therapy. They would insert human genes into a bacteria cell. Then the bacteria cell would transcribe and translate the information into a protein Then they would introduce the protein into human cells. However today, most gene therapy studies are aimed at cancer and hereditary diseases linked to a genetic defect.

The First Approach

The First Time Ever

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. This only worked for a few months .

How It Works

A vector delivers the therapeutic gene into a patients target cell. The target cells become infected with the viral vector. The vectors genetic material is inserted into the target cell. Functional proteins are created from the therapeutic gene causing the cell to return to a normal state.

How it actually happens

 Germ line gene therapy :In the case of germ line gene therapy, Germ cells are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. Somatic gene therapy :In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.

Types Of Gene Therapy

Viruses: A Vector
These replicate by inserting their DNA into a host cell. Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait. Four different types: - Retroviruses - Adenoviruses -Adeno-associated Viruses -Herpes Simplex Viruses

o The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. o When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. Reverse transcriptase is the enzyme carried with the virus ,through which this process is carried out. Integrase is the enzyme carried in the virus which helps in the insertion of DNA copy produced in the chromosomes. o Now the host cell has been modified to contain new genes. If this host cell divides later, its descendants will all contain the new genes. o One of the problems is that Integrase inserts the gene anywhere randomly because it has no specific site. . o If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur.

Retroviruses

Adenoviruses

Carries their genetic material in the form of double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans. The inserted DNA is not incorporated into the host cell's genetic material. The extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. So it again has to be reinserted when more cells divide. This vector system has been promoted for treating cancer and indeed the first gene therapy product to be licensed to treat cancer.

 Adeno-associated Virus is small, has single stranded DNA that insert genetic material at a specific point on chromosome 19. It belongs to parvovirus family. It causes no known disease and doesn't trigger patient immune response. It has got low information capacity The gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. It includes hemophilia treatments. For example, a genecarrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.

Adeno-associated Viruses

Herpes Simplex Viruses

The Herpes simplex virus is mostly examined for gene transfer in the nervous system. The double stranded DNA viruses that infect neurons. For Example: Herpes simplex virus type 1. Antibodies to HSV-1 are common in humans, however complications due to herpes infection are somewhat rare.

Direct injection of DNA simplest method of non-viral transfection. Requires a lot of DNA. Cellular uptake is inefficient. Physical Methods to Enhance Delivery Electroporation : - uses short pulses of high voltage to carry DNA across the cell membrane. - temporary formation of pores in the cell membrane is caused, allowing DNA molecules to pass through. - efficient and works across a broad range of cell types ,but usage has been limited. -More recently a newer method of electroporation, termed electronavalanche transfection, has been used in gene therapy experiments. Gene Gun: -DNA is coated with gold particles and loaded into a device which generates a force to achieve penetration of DNA/gold into the cells. Sonoporation: -uses ultrasonic frequencies to deliver DNA into cells. Magnetofection: -DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.

Non-viral Options

Chemical Methods to Enhance Delivery

1.

2.

Oligonucleotides: it inactivates the genes involved in the disease process. It includes several strategies like, use of antisense specific to the target gene to disrupt the transcription of the faulty gene or use of small molecules of RNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA. Lipoplexes and polyplexes: Its most common use has been in gene transfer into cancer cells. lipoplexes are useful in transfecting respiratory epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis. Complexes of polymers with DNA are called polyplexes. consist of cationic polymers and production is regulated by ionic interactions. Polyplexes cannot release their DNA load into the cytoplasm.

Hybrid methods

Hybrid methods developed combined two or more techniques. Virosomes are one example; they combine liposomes with an inactivated HIV or influenza virus.

Major developments in gene therapy

1972: Friedmann and Roblin authored a paper in Science

titled "Gene therapy for human genetic disease? 1990: The first approved gene therapy case in the United States took place on September 14, 1990 on a four year old girl named Ashanti DeSilva. 1992: Doctor Claudio Bordignon performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases. 1999: Death of Jesse Gelsinger in a gene-therapy experiment resulted in a significant setback to gene therapy research in the United States.

 2003 :University of California research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol. It is a significant achievement because viral vectors are too big to get across the bloodbrain barrier. 2006 : Scientists at the National Institutes of Health have successfully treated metastatic melanoma in two patients using killer T cells which demonstrated that gene therapy can be effective in treating cancer. In March ,an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. In May, a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from Telethon Institute for Gene Therapy (HSR-TIGET) developed a way to prevent the immune system from rejecting a newly delivered gene.

 In November ,Preston Nix from the University of Pennsylvania School of Medicine reported, a gene-based immunotherapy for the treatment of HIV that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope. The insertion of T cells (genetically modified) was safe and well tolerated. 2007 :On 1 May, Moorfields Eye Hospital and Institute of Ophthalmology in London, announced the world's first gene therapy trial for inherited retinal disease which was carried out on a 23 yearold British male. 2009 :In September, researchers at the University of Washington and University of Florida were able to give trichromatic vision to squirrel monkeys using gene therapy, a hopeful precursor to a treatment for color blindness in humans.

 2011: In 2007 and 2008, a man was cured of HIV by repeated Hematopoietic stem cell transplantation with double-delta-32 mutation this cure was not completely accepted by the medical community until 2011.

Problems and ethics

Short-lived nature of gene therapy Immune response Problems with viral vectors Multigene disorders Chance of inducing a tumor (insertional mutagenesis) Deaths have occurred due to gene therapy

Recent Developments
Genes get into brain using liposomes coated in polymer call polyethylene glycol. Creation of tiny liposomes that can carry therapeutic DNA through pores of nuclear membrane Sickle cell successfully treated in mice

Thank You for the patience and cooperation Special References: WIKIPEDIA

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