Gene Therapy

Pramod kumarM.Sc MBT(final)

MDU Rohtak

Genes

• Are carried on a chromosome

• The basic unit of heredity

• Encode how to make a protein.

• DNA RNA proteins

• Proteins carry out most of life’s function.

• When altered causes dysfunction of a protein

What is Gene Therapy It is a technique for correcting defective

genes that are responsible for disease development

There are four approaches:1. A normal gene inserted to compensate for a

nonfunctional gene.2. An abnormal gene treated for a normal gene3. An abnormal gene repaired through selective

reverse mutation4. Change the regulation of gene pairs

The Beginning…

• 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

The First Case

• 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.

How It Works

• A vector delivers the therapeutic gene into a patient’s target cell

• The target cells become infected with the viral vector

• The vector’s 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

Choices of Vectors

The ideal vector system would have the following characteristics:

(1) an adequate carrying capacity; (2) to be undetectable by the immune system; (3) to be non-inflammatory. (4) to have long duration of expression and/or the ability to

be safely re-administered.

• Viral vectors:• Retrovirus• Adenovirus• Adeno-associated virus• Herpes Simplex Virus

Non-viral vectors:LiposomeDNA–polymer conjugatesNaked DNA

Viruses

• 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• Created double stranded DNA copies from RNA genome

– The retrovirus goes through reverse transcription using reverse transcriptase and RNA

– the double stranded viral genome integrates into the human genome using integrase• integrase inserts the gene anywhere because it has no

specific site• May cause insertional mutagenesis

– One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division)

– vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety

Adenoviruses

• Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans

• The inserted DNA is not incorporate into genome

• Not replicated though– Has to be reinserted when more cells divide

• Ex. Common cold

Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

Adeno-associated Viruses Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific

point on chromosome 19

From parvovirus family- causes no known disease and doesn't trigger patient immune

response.

Low information capacity

gene is always "on" so the protein is always being expressed, possibly even in instances

when it isn't needed.

hemophilia treatments, for example, a gene-carrying vector could be injected into a

muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.

Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed

Factor IX injections for more than a year

Non-viral Options• Direct introduction of therapeutic DNA

– But only with certain tissue– Requires a lot of DNA

• Creation of artificial lipid sphere with aqueous core, liposome Carries therapeutic DNA through membrane Chemically linking DNA to molecule that will bind to special cell receptors

– DNA is engulfed by cell membrane– Less effective.

Problems

• Delivery of DNA• Achieving high level expression• Maintaining stable expression• Tissue-specific expression• in vivo regulation

Gene therapy

In vivo Ex vivo

in vivo and ex vivo schemes

IN VIVO

EX VIVO

Ex-vivo gene therapy

Usually ex-vivo gene therapy involves the following procedure.

1.Collect cells from an affected individual.2.Correct the genetic defect by gene transfer into the isolated cells.3.Select and grow the genetically corrected cells.4.Either infuse or transplant them back into the patient.

How to fix it

AB C a beneficial geneA

virus modified virus

• A virus is found which replicates by inserting its genes into the host cell's genome. This virus has three genes - A, B and C.

• Gene A encodes a protein which allows this virus to insert itself into the host's genome.

• Genes B and C actually cause the disease this virus is associated with Replace B and C with a beneficial gene. Thus, the modified virus could introduce your 'good gene' into the host cell's genome without causing any disease.

• So we use the modified virus to fix the “broken window”

Ex-vivo gene therapy

First gene therapy trial

for ADA deficiency -

1990

In- vivo gene therapy

In vivo gene therapy entails the direct delivery of a remedial gene into the cells

Of a particular tissue of a prospective patient.

Retroviral vectors require that the target cells must be dividing in order to be

Infected.

A no. of strategies which include using viral and non-viral vector system to deliver

a therapeutic gene to cells of the target tissue, have been devised.

Picture

http://encarta.msn.com/media_461561269/Gene_Therapy.html

In Vivo Gene Therapy for Cystic Fibrosis

• Cystic fibrosis is a heterogeneous recessive genetic disorder with features that reflect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

• Cystic fibrosis was first described as a disease in the late 1930s by Dorothy Hansine Andersen. In 1988, the first mutation for CF, ΔF508, was discovered by Francis Collins, Lap-Chee Tsui and John R. Riordan on the 7th chromosome of the human genome

• The mutation for CF on the 7th chromosome

• Classic cystic fibrosis is characterized by chronic bacterial infection of the airways and sinuses, fat maldigestion due to pancreatic exocrine insufficiency, infertility in males due to obstructive azoospermia, and elevated concentrations of chloride in sweat.

• Patients with non classic cystic fibrosis have at least one copy of a mutant gene that confers partial function of the CFTR protein, and such patients usually have no overt signs of maldigestion because some pancreatic exocrine function is preserved.

Hallmarks of CF • Very salty-tasting skin • Appetite, but poor

growth & weight gain• Coughing, wheezing &

shortness of breath • Lung infectionsLung infections, e.g.

pneumonia/bronchitis

Treatment

Gastrointestinal Treatment • Modified diet

Due to pancreatic disorders, children with CF require a modified diet, including vitamin supplements (vitamins A, D, E, and K) and pancreatic enzymes. Maintaining adequate nutrition is essential. The diet calls for a high-caloric content (twice what is considered normal for the child's age), which is typically low in fat and high in protein. Patients or their caregivers should consult with their health care providers to determine the most appropriate diet.

•The only way to cure CF would be to use gene therapy to replace the defective gene or to give the patient the normal form of the protein before symptoms cause permanent damage. •The major goal in treating CF is to clear the abnormal and excess secretions and control infections in the lungs, and to prevent obstruction in the intestines.

•For patients with advanced stages of the disease, a lung transplant operation may be necessary. •Although treating the symptoms does not cure the disease, it can greatly improve the quality of life for most patients and has, over the years, increased the average life span of CF patients to 30 years.

Gene Therapy for Cystic Fibrosis

• Cystic fibrosis should be an ideal candidate for gene therapy, for four main reasons:

• (1) it is a single gene defect; • (2) it is a recessive condition, with heterozygotes

being phenotypically normal (suggesting gene dosage effects are not critical);

• (3) the main pathology is in the lung, which is accessible for treatment;

• (4) it is a progressive disease with a virtually normal phenotype at birth, offering a therapeutic window.

Cystic Fibrosis

Protein Function and Biochemistry

• CFTR controls chloride ion movement in and out of the cell.

Protein Function Continued

• In the case of CF, gene therapy involves inhaling a spray that delivers normal DNA to the lungs.

• The goal is to replace the defective CF gene in the lungs to cure CF or slow the progression of the disease.

Problems with Gene Therapy• 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• May induce a tumor if integrated in a tumor suppressor gene because

insertional mutagenesis

References• Molecular biotechnology principles and applications of recombinant DNA, Bernard

R.Glick and Jack J.Pasternak 2nd edition. Page no. 560, 566.

• Burdette, Walter J. The Basis for Gene Therapy. Springfield: Charles C Thomas,2001.

• Crayton, Stephanie. “First Clinical Trial Of Gene Therapy For Muscular Dystrophy Now Under Way.” Medical News Today. 1 April 2006. University of North Carolina at Chapel Hill. 11 November 2006 <www.medicalnewstoday.com>.

• Gene Therapy. Human Genome Project Information. 18 November 2005. U.S. Department of Energy Office of Science, Office of Biological and Environmental Research, Human Genome Program. 12 September 2006 <http://www.ornl.gov/hgmis>.

• Peel, David. “Virus Vectors & Gene Therapy: Problems,Promises & Prospects.” Virus Vectors & Gene Therapy. 1998. Department of Microbiology & Immunology, University of Leicester. 11 November 2006 <http://www.tulane.edu/~dmsander/WWW/335/peel/peel2.html>.