1. ZEESHAN GAUHAR PhD SCHOLOR-BIOTECHNOLOGY
Human Gene Therapy
ZEESHAN GAUHAR
PhD SCHOLOR-BIOTECHNOLOGY

2. contents GENE THERAPY APPROACHES HISTORY TYPES GERMLINE
SOMATIC GENE THERAPY
VECTORS
VIRAL
NON VIRAL
DISADVANTAGES
GENE THERAPY IN SICKLE CELL ANEMIA
ETHICAL AND SOCIAL CONSIDERATION
CONCLUSION

3. GENE THERAPY
Gene therapy is the use of genes as medicines, basically to correct defective genes responsible for genetic disorder.

4. APPROACHES
A normal gene could be inserted into a nonspecific location within the genome to replace the Nonfunctional gene (most common)
An abnormal gene could be swapped for a normal gene homologous recombination
An abnormal gene could be repaired through selective reverse mutation
Regulation (degree to which a gene is turned on or off) of a particular gene could be altered

5. Historical Perspectives
1990, first approved gene therapy case in the United States took place on a four year old girl for the treatment of ADA- SCID, a severe immune system deficiency. The effects were only temporary, but successful.
1992, Claudio Bordignon of Italy performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary disease.
1993, a new born baby Andrew Gobea, with SCID, was treated by gene therapy technique using retrovirus vector carrying ADA gene.
1999, gene therapy suffered a major setback with the death of 18 year old Jesse Gelsinger who participated in a gene therapy trial for ornithine transcarboxylase deficiency.

6. 2003, “FOOD and DRUG ADMINISTRATION” (FDA) placed a temporary halt on all gene therapy trials using retrovirus vector in blood stem cells.
Then in April 2003, FDA eased the ban after regulatory review of the protocol in USA, UK, France, Italy and Germany.
2003, Los Angeles research team inserted genes into brain using liposome coated in a polymer called polyethylene glycol.

7. In 2005, scientists were able to repair deafness in guinea pig by using adenovirus vector.
In 2006 (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 2007, a team of British doctors from Moorefield’s Eye Hospital and University college of London, announced the world’s first gene therapy trial to test a revolutionary gene therapy treatment for a type of inherited retinal disease.

8. In 2009 (March), the School of Pharmacy in London tried nanotechnology based gene therapy to target and destroy hard-to-reach cancer cells.
In 2010, a paper by Komaromy et al. report gene therapy for a form of achromatopsia (complete colour blindness) in dogs.
In 2012, Glybera became the first gene therapy treatment to be approved for clinical use in either Europe or the United States after its endorsement by the European Commission.

9. TYPES OF GENE THERAPY

10. 1. Germ line gene therapy: germ cells (sperm or egg) are modified by the introduction of functional genes, which are integrated into their genome. 2. Somatic gene therapy: therapeutic genes are transferred into the somatic cells of a patient.

15. VECTORS Two types of vectors are used, Viral Non viral
Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to harness their ability by manipulating the viral genome to remove disease causing genes and insert theurapatic ones.

16. Retrovirus RNA viruses
Use integrase to integrate its genome into the host genome
Problems
integrase enzyme can cause insertional mutagenesis i.e. can insert genetic material of the virus into any arbitrary position in the genome of the host.
successful application till date
X linked severe combined immune deficiency
SCID due to ADA deficiency with relative success.
Cystic fibrosis

17. Adeno-associated viruses (AAVs)
single stranded DNA viruses
can infects a broad range of cells
Can insert genetic material at a specific site on chromosome19 with near 100% certainty
Drawbacks
A small virus, carrying only 2 genes in its natural state
can produce unintended genetic damage because the virus inserts its genes directly into host cell’s DNA

18. Herpes simplex virus (HSV)
Double stranded DNA viruses
human neurotropic virus
large genome which enable scientist to insert more than one therapeutic gene into a single virus
HSV makes an ideal vector as it can infect a wide range of tissues including muscle, liver, pancreas, and nerve and lung cells.

19. Adenovirus Double stranded DNA viruses
DNA molecule is left free in the nucleus of the host cell, and transcribed just like any other gene
can infect a broader variety of cells
Applications
Gendicine,first gene therapy product to be licensed to treat head and neck cancer

21. Non-Viral Methods
electroporation (creation of electric field induced pores in plasma membrane)
sonoporation (ultrasonic frequencies to disrupt cell membrane),
magnetofection (use of magnetic particle complexed with DNA),
gene guns (shoots DNA coated gold particles into cells by using high pressure)
receptor mediated gene transfer

22. chemical methods
use of synthetic oligonucleotides (to inactivate defective genes by using antisense specific to target gene)
lipoplexes (made up of anionic and neutral lipids)
polyplexes (complex of polymers with DNA)
Hybrid methods e.g vibrosomes that combine liposomes with an inactivated HIV or influenza virus and viral vectors with cationic lipids

23. Disadvantages of Gene Therapy
Short-lived nature of gene therapy
Immune response
Problem with viral vectors
Multigenic disorders
Insertional mutagenesis

24. Gene therapy in sickle cell anemia
caused by a change in just one amino acid at a specific site in the β-globin gene.
results in the production of sickle shaped cells which prevent oxygenated blood from flowing through.

25. the first sickle-cell gene therapy to be tested in humans.
INJECTING GENES
the first sickle-cell gene therapy to be tested in humans.
At the regenerative medicine and stem cell research centre of the University of California, Los Angeles, molecular geneticist and physician Donald Kohn is developing protocols for a clinical trial of this technique that is due to start enrolling patients by the end of 2014.

26. first harvest bone marrow from the hip bones of patients with sickle-cell disease
then extract haematopoietic stem cells from the marrow
Using a viral vector,insert a new, working haemoglobin gene into the cells’ DNA
the old, faulty haemoglobin gene will still be present, but it will go silent as the new gene takes over
The modified cells will then be infused back into the patient’s bloodstream
They will migrate to the bone marrow, where they can provide a continual source of healthy red blood cells.

27. This technique has been tested by injecting modified human haematopoietic stem cells into mice, and found that they were free of sickle cells 2 to 3 months later.(Romero, Z. et al. J. Clin. Invest. 123, 3317– 3330 (2013).

28. challenges
researchers must harvest the bone marrow itself, which can be difficult and slow, and limits the number of cells that can be collected at one time

29. GENE EDITING Zinc finger nucleases(ZFNs)
Further away from clinical trials, but potentially a lot more exciting, is gene editing.
Zinc finger nucleases(ZFNs)
Transcription activator-like effector nuclease(TALEN)
CRISPRs (clustered regularly interspaced short palindromic repeats)

30. Zinc-finger nucleases (ZFNs)
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain.
engineered zinc finger nucleases (ZFNs) bind to a specific section of DNA and create a break at both ends
Cells will start to repair the break, at which point a specific sequence of laboratory-made DNA can be slotted into the gap.

31. Transcription activator-like effector nuclease TALEN
Transcription activator-like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain.
Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any desired DNA sequence. By combining such an engineered TALE with a DNA cleavage domain (which cuts DNA strands), one can engineer restriction enzymes that are specific for any desired DNA sequence.

32. clustered regularly interspaced short palindromic repeats(CRISPR)
DNA loci containing short repetitions of base sequences.
CRISPRs are often associated with cas genes that code for proteins related to CRISPRs.
used for gene editing since 2013.

33. Ethical and Social Consideration
Some of the ethical considerations for gene therapy include:
Deciding what is normal and what is a disability
Deciding whether disabilities are diseases and whether they should be cured
Deciding whether searching for a cure demeans the live of people who have disabilities
Deciding whether somatic gene therapy is more or less ethical than germ line gene therapy

34. Conclusion
scientists believe that after 20 years, this will be the last cure of every genetic disease. Genes may ultimately be used as medicine and given as simple intravenous injection of gene transfer vehicle that will seek our target cells for stable, site-specific chromosomal integration and subsequent gene expression. And now that a draft of the human genome map is complete, research is focusing on the function of each gene and the role of the faulty gene play in disease. Gene therapy will ultimately change our lives forever.

35. References
Human Gene Therapy : A Brief Overview of the Genetic Revolution Sanjukta Misra*
Romero, Z. et al. J. Clin. Invest. 123, 3317–3330 (2013).
Fine, E. J. et al. Nucleic Acids Res. 42, e42 (2014).
Suzuki, K. et al. Cell Stem Cell 15, 31–36 (2014).

36. THANKS