A complete elaborate presentation on research papers from the SCIENCE journal. Help give a brief insight into the disease, genetics behind it, and proposed treatment for it.

1. Published on: 03.01.2008
Journal: SCIENCE
Impact factor: 37.205
Anjali Mandal-BT17008
Dipti Mundhe-BT17010
B.Tech. Semester-4

2. What is ‘Sickle cell anaemia’?
• Sickle cell anaemia (SCA) is a genetic blood disorder where the
normal, biconcave shaped RBC’s change to ‘sickle shaped’ cells.
Normal RBC’s
Sickle shaped RBC’s

3. What is Sickle Cell Anemia?
• It is caused by the mutation in the “hemoglobin beta gene (HBB)”
present on chromosome 11.
• It is a recessive disease, which means that for a person to have sickle
cell anemia, both the copies of the gene must be mutated.
• If an individual just carrier’s one copy of this mutated gene then
he/she is known to be a carrier of the disease.

4. What causes ‘Sickle cell anaemia’?
• A point mutation in the DNA, changes the Adenine (A) to Thymine (T),
which changes the so translated protein, i.e. Glutamate to Valine.
• This conversion of glutamate to
valine causes the hemoglobin
molecules to form sticky ends.
• This produces long fibers of hb.
Thus changing the shape of
RBCs.
• This forms hBS that is the
mutated copy.

5. Global impact of the disease
North America: 1.4%
South America: 1.4%
Africa: 76%
Asia: 19.6%

6. Impact in India

7. Traits of Sickle cell anaemia
• Low hb levels
• Episode of pain
• Swelling of hands and feet
• Frequent infection
• Difficulty in breathing
• Higher risk of stroke
• Pulmonary hypertension

8. Current treatments available...
• The current treatments for SCA is treating the symptoms as and when
they appear. Like:
• Avoiding painful episodes- use hydroxyurea
• Avoiding body pain- over the counter analgesics (ibuprofen)
• Treating anaemia- intake of folic acid, in severe cases regular blood
transfusion
• Treating infections- antibiotics like penicillin

9. Upcoming approaches:
• Bone marrow transplants
• Stem cell therapy combined with gene therapy

10. Eureka moment!
After the Shinaya Yamanaka published
his research on iPSC, J Hanna exploited
it’s further potential by using this
technique to find a cure for SCA.

11. Aim of the experiment
• The aim of the experiment was to convert somatic cells (tail-tip
fibroblast) to pluripotent cells.
• MTF- Mouse tail-tip fibroblast
MTF iPSC
Oct4-Neo, Klf4, c-Myc, Sox-2
iPSC- induced
pluripotent stem cells

12. Continued…
• Then by homologous recombination, replace the defected gene with
a healthy copy of it.
Diseased copy
Healthy copy
*homologous recombination: is a type
of genetic recombination in
which nucleotide sequences are exchanged
between two similar or identical molecules
of DNA.

13. Continued…
• Hence recovering the normal functional aspects of RBC’s.

14. First step: Obtain iPSC!
• Tail-tip fibroblast (TTF) from 12 week humanized knock-out mice was
harvested. (hBs|hBs mouse)
• These TTF cells were infected with retroviruses containing 4
transcription factors, i.e. oct-4-neo, 4-Klf, Sox-2, c-Myc.
• The mice used were tagged with neomycin resistance gene ‘oct-4-
neo’
Oct-4 Promoter Neomycin resistance

15. Obtaining iPSC
• Oct-4 is the “pluripotency regulator of the cell.”
• The cells treated with 4 transcription factors are checked for
pluripotency by the expressing of ‘oct-4-neo’i.e. their resistance to
neomycin
• The further 24 neomycin resistant colonies were picked up at day 16.
• These were designated a cell-line name- ITTO26.

16. Check-time
ES CELLS
AP, SSEA1, Nanog AP, SSEA1, Nanog
ITTO26 CELLS
Both the control, i.e. mouse embryonic cells and induced pluripotent stem cells
were checked for the expression of stemness markers viz. AP, SSEA1, Nanog

17. Hematopoietic Differentiation
• The ITTO26 cell line and the embryonic stem cells were infected with
‘Moloney virus’.
• This virus carried the HoxB4* protein tagged with GFP.
• They were cultured on a media containing hematopoietic cytokines to
support hematopoietic differentiation.
*HoxB4 protein- it is generally expressed in homeodomain cells or the hematopoietic
progenitors

18. Confirmation for hemopoietic differentiation
• To confirm for hematopoietic differentiation these cells were run on a
fluorescent activated cell sorter (FACS), stained with two antibodies:
CD41
c-kit antigen
• These are the two early markers of hemopoietic progenitor cells as
well as markers for erythroid differentiation.

19. What is FACS?
• Fluorescence-activated cell sorting (FACS) is a specialized type of flow
cytometry. It provides a method for sorting a heterogeneous mixture
of biological cells into two or more containers, one cell at a time,
based upon the specific light scattering and fluorescent
characteristics of each cell.

20. What does a typical FACS graph look like?

21. Verification of hemopoietic differentiation
*V6.5 ES- Embryonic Stem
cells (control)
ITTO25 iPS- Induced
pluripotent stem cells.

22. Survival rate check
Embryonic cells
ITTO26 iPSC
No.ofcells
No. of days
The next check for
stem cell like
behavior is the
survival rate of the
obtained iPSC.

23. Next step: Gene Correction!
• ITTO26 cells were then specifically corrected for their gene defect by
homologous recombination.
• The cells were electroporated and human hBA wild type gene.
• These cells were treated with hygromycin and gancylovir to check for
correct gene targeting.
• 1 colony out of the 72 colonies were identified as correctly gene
targeted colony.
• This was checked by performing PCR and further digestion by Bsu36I.

24. Check for recombination
• The Bsu36I is a restriction
enzyme which is site specific,
therefore its only able to cut
the sites containing hBA
• This proves the correct
homologous recombinant
alleles.

25. Post successful recombination
• The cells with corrected gene was engrafted to three mice suffering
from sickle cell anemia (hBS|hBS)
• 12 weeks post transplants the engraftment was stable
• This was checked by presence of GFP in peripheral blood
• The DNA samples of these mice on electrophoresis showed the
presence of human globin A and S.
• Also the quantity of human globin A increased after 8 weeks.

26. Parameters to test for:
• A control group of mice suffering from sickle cell anemia was used to
compare the results of engrafted mice.
• These were the parameters that was checked for:
Morphology
Complete blood profiling
Urine concentration
Weight
Breathing rate

27. Morphological check

28. Complete blood profiling

29. Some more findings

30. Conclusion
• The mice having sickle cell anemia improved in certain aspects:
Urine concentration defect resolved
Breathing improved
Blood hemoglobin levels recovered
RBCs count improved

31. Drawbacks
• The implanted iPSC still have the risk of tumor formations
• The study was only monitored for a short term period of about 3
months.