THE SUPERBOY

1. THESUPERBOY (MUSCLE HYPERTROPHY) BSB 3143 GENE TECHNOLOGY KALAISELVI MOHANRAJ SB09031 RUBINI DEVI SELVARAJOO SB09005 SUMITHALAKSMY GUNASEGARAN SB09063

2. INTRODUCTION BOSTON — Somewhere in Germany is a baby Superman, born in Berlin with bulging arm and leg muscles. Not yet 5 in 2004, he can hold seven-pound weights with arms extended, something many adults cannot do. He has muscles twice the size of other kids his age and half their body fat. The Associated Press, 2011

3. PROBLEM STATEMENT According to a DNA test, the boy has a genetic mutation that boosts muscle growth. This genetic mutation is called the myostatin deficiency. The boy’s condition is the result of carrying 2 mutated copies of the myostatin gene. His mother has only 1 such mutation. The Associated Press, 2011

4. Gene Therapy • Main steps: • Identification and • characterization of gene • Cloning of gene • Choice of vector • Method of delivery • Expression of gene

5. Identification & characterization Of Gene • The first step is to identify the genetic defect and to clone a good copy of the gene involved. • Gene Knockout (Knock out mouse) • Isolate embryonic stem cells that originated from male brown mice with a normal MSTN gene (blue) • A copy containing a mutated (inactive) MSTN gene , and a drug resistance marker gene is combined.

6. Homologous recombination • - Similar genes will swap. • - The mutant MSTN gene plus drug resistance marker gene is now incorporated into the genome and the normal version is kicked out. • Add drug • - Cells that haven’t incorporated the inactive MSTN gene don’t have the drug resistant marker gene. • Adding the drug kills cells without the marker, leaves us with a clean batch of cells that all have an inactive version of the MSTN gene. • Grow Chimeric Mice • - Transplant the stem cells that carry the inactive MSTN gene into a white mouse embryo to create chimera.

7. Chimeras have patches of cells throughout their bodies that grew from white mouse cells and patches that grew from brown stem cells. • Some of the cells that have the inactive MSTN gene may develop into reproductive cells. • -Chimeras are easy to identify because they have both brown and white patches of fur. • Mate Male Chimera • - If a male chimera has some reproductive cells (sperm) that originated from the brown stem cells, he will produce some brown offspring when mated with a white female.

8. Test & Breed Brown Offspring • -Half of the brown offspring will have copy of the inactive MSTN gene in all of their cells – including their reproductive cells. These mice have one normal copy of the MSTN gene and one inactive copy. • - These mice can be identified by PCR and then bred with each other. • One fourth of the resulting offspring will have two copies of the knocked out or inactive MSTN genes.

9. Cloning of gene

10. Choice Of Vector • Adeno-associated virus (AAV) is a small virus which • infects humans and some other primate species. • AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

11. Advantages: • AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.  • It is possible to target specific cell types by engineering proteins on the virus surface to recognize special proteins on the target cell’s surface. • 95% of the time, it will integrate into a specific region on chromosome 2, greatly reducing the chance that integration will disrupt the function of • other genes in the cell. • - Does not cause immune response.

12. Method of Delivery • Ex-vivo Gene Therapy • cells are first cultured or synthesized outside of an organism and then inserted into the organism to provide the treatment • Steps: • Apheresis • Purification • Genetic Modification • Expansion • Infusion

14. Gene Expression Expression System: Finally, the gene is expressed in the patient.

17. Conclusion • Gene therapy is a powerful new technology that still requires several years before it will make a noticeable impact on the treatment of disease. • Several major deficiencies include poor delivery system, both viral and non viral and poor gene expression after genes are delivered. • However, in every field of medicine, the ability to give the patient therapeutic genes offers extraordinary opportunities to treat, cure and ultimately prevent a vast range of diseases that now plague mankind.