The document discusses gene therapy, a technique for correcting defective genes responsible for diseases, outlining its history, methods, and various vector types used for gene delivery. Key approaches include inserting normal genes, repairing abnormal ones, and changing gene regulation, with notable applications in conditions like cystic fibrosis and severe combined immunodeficiency. Challenges include effective delivery, stable expression, and potential immunological responses, with current research focusing on improving these aspects for better treatment outcomes.

1. Gene Therapy
Pramod kumar
M.Sc MBT(final)
MDU Rohtak

2. 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

3. 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 gene
3. An abnormal gene repaired through selective
reverse mutation
4. Change the regulation of gene pairs

4. 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

5. 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.

6. 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

7. 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:
Liposome
DNA–polymer conjugates
Naked DNA

8. 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

9. 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

10. 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

11. 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.

12. 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

13. 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.

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

15. Gene therapy
In vivo Ex vivo

16. in vivo and ex vivo schemes
IN VIVO
EX VIVO

17. 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.

18. How to fix it
A
B 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”

19. Ex-vivo gene therapy

20. First gene
therapy
trial for
ADA
deficiency
-1990

22. 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.

23. Picture 
http://encarta.msn.com/media_461561269/Gene_Therapy.html

25. In Vivo Gene Therapy for
Cystic Fibrosis

26. • 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.

27. 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

28. 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.

29. 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

30. Protein Function and Biochemistry
• CFTR controls
chloride ion
movement in
and out of the
cell.

31. Protein Function Continued

32. • 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.

33. 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

34. 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>.