Gene Therapy

Introduction

Pages 1-11

Gene Therapy

Purpose:– Introduce a functional gene into a person who

lacks this gene– Gene therapy is potentially useful for treating

recessive diseases– Parents are heterozygotes– Patients lack a particular enzyme– Gene therapy is used to replace the missing

enzyme

Gene therapy

The first disease that gene therapy was used for was Severe Combined Immune Deficiency (SCID)

In this disease, there is a lack of the enzyme adenosine deaminase (ADA)

Gene therapy is used to give a patient a functional ADA gene

Introduce the gene into bone marrow cells and the patient can then produce WBCs throughout their life and produce the enzyme

What diseases should gene therapy be used for?

Diseases must result from the defect in a single gene

Gene must have been identified and purified Cells affected by the gene defect must be

accessible for gene therapy– Problems in treating neurological disease– Must get past the blood brain barrier

Diseases chosen usually have few effective treatments

What cells should be targeted to be altered?

Gene therapy is for altering somatic or body cells and not germ cells

This treatment will be used to correct a defect in an individual but the correction is NOT passed on to subsequent generations

Gene therapy is used to treat diseases and not alter characteristics of the next generation

Transcription and Translation

DNA codes for the gene that you want to insert– Insert the gene for ADA to treat SCID

If gene therapy is successful, then the ADA enzyme will be made

SCID - ADA Lung cancer - p53 tumor suppressor gene Cystic fibrosis - Cystis fibrosistransmembrane

conductance regulator (CFTR) Leukemia - Interleukin 2 (IL-2)

Look at table 2, pages 5-7

Different diseases can be treatedby introducing different genes

How do you make the recombinant DNA?

Vector– Plasmid– Place to incorporate the foreign DNA

The DNA that codes for the missing enzyme

– A marker gene - antibiotic resistance gene Determine if the vector entered the cell

Foreign DNA

Steps in making recombinant DNA

Need DNA from 2 different sources– In this class, we will transfer DNA from one

plasmid and put it into another plasmid Cut DNA with restriction enzymes

– Create staggered or blunt end cuts Match the DNA from the 2 different

sources and let them combine DNA ligase seals the 2 pieces together

DNA structure

When performing cloning, how do you find the gene you are interested in?

Get a gene library– Collection of different genes

Use a PROBE to find your gene in this gene library

A probe is complementary to your gene of interest

How is the PROBE used?

Gene sequence = AGCTGGAC Probe = TCGACCTG

Heat is used to separate the two strands Probe is added to the gene sequence Probe is labeled at one end with a

radioisotope so it can be detected if it binds to the gene

How is the PROBE used?

Locate a functional ADA probe to perform gene therapy for SCID

Use a gene library from someone who DOES NOT have SCID

Use a probe specific for the ADA sequence Isolate the DNA to be used in gene therapy

Probes are also used in Southern Blots, Northern Blots and Western Blots