2. What is Diabetes Mellitus?
• Diabetes is a chronic disease in which
the body cannot regulate the amount
of sugar in the blood.
• High blood sugar
• This is because either:
• Their pancreas does not make enough insulin
• Their cells do not respond to insulin normally
• Both of the above
3. Types of Diabetes
• In Type 1 diabetes, the pancreas cells ,
called islets of Langerhans,
that make insulin stop working.
Daily injections of insulin are needed.
The exact cause is unknown.
• Type 2 diabetes is more common and
in this case, the body makes
insulin. But either their pancreas does
not make enough insulin or the body
cannot use the insulin well enough.
5. Problem
Normally, Type 1 diabetes patients who have received islet cell
transplants must be treated regularly with immunosuppresive
treatments so that the body does not attack these new cells. But this is
also unsafe because it puts the patient at risk of contracting other
diseases if his immune system is suppresed.
6. Hypothesis
I will use gene therapy to use a vector to add an
immunosuppresive gene to the cells so that the bodies immune system
will not attack the newly implanted cells and there will no need for
alternate immunosuppresive treatment. If the transplant is succesfully
accepted by the bodies immune system, it will allow for natural,
endogenous insulin production to occur.
7. What is Gene Therapy?
• It is the transfer of genetic
material into cells or tissue
to prevent or cure a disease.
• For this proposal, I will use
in-vitro gene therapy which is
based on the transfer of the
vector carrying the therapeutic
gene into cultured cells from
the patient.
8. What is a Viral Vector?
• Viral vectors are a tool commonly
used by molecular biologists to
deliver genetic material into cells.
This process can be performed inside
a living organism (''in vivo'') or in
cell culture (''in vitro'').
• Viruses have evolved specialized
• molecular mechanisms to efficiently transport their genomes inside
the cells they infect. Delivery of genes by a virus is termed
transduction and the infected cells are described as transduced.
9. What is a Viral Vector?
• Viral vectors are tailored to their specific applications but generally share a few
key properties:
• Safety – they are involved in deletion of the viral genome critical for viral
replication. They can infect cells but do not replicate themselves.
• Low toxicity – they have minimal effect on the physiology of the cell they
infect
• Stability – they are genetically stable so they cannot rearrange their genome
as most viruses do
• Cell type specificity – they can be modified to affect a specific kind of cell
• Identification – they are given genes (markers, like antibiotic resistance) to
help identify which cells took up the viral genes
10. Why Adeno-Associated Virus?
• The unique life cycle of adeno-associated virus (AAV) and its ability to
infect both nondividing and dividing cells with persistent expression
have made it an attractive vector. An additional attractive feature is
its lack of apparent pathogenicity.
• Gene transfer studies using AAV have shown significant progress at
the level of animal models.
• AAV vectors are derived from AAV type2 virus, which is a non-
pathogenic, replication-defective parvovirus, and can integrate into
host genome at a specific locus.
11. Gene
• I will use the CD47 gene which produces CD47 protein.
• This protein acts as a passport that tells your immune system not to
attack a cell.
• Cancer cells produce this protein in excess and it is what protects
them from being destroyed by the immune system.
• It is possible that by implementing this gene, it will help the newly
implanted islet cells to avoid being attacked by the immune system.
12. What Is Needed For Gene Therapy
Gene Vector Target Cell Routes of
Administration
Animal Models
CD47 Gene Adeno-
Associated Virus
Islet Cells Injection into
the pancreas
AKITA Mice
13. Experiment
• I will use AKITA mice who have type 1 diabetes as my animal model.
• I will use two groups of 20 mice each:
• Group A will receive the islet cell transplants that carry de CD47
gene.
• Group B will receive islet cell transplants without the added
gene.
14. Methodology
• 1. I have identified the gene (CD47) that I will insert into the viral
vector.
• 2. I have identified the right regulatory sequence, or promoters, that
are positioned in front of the gene to be expressed.
• 3. I extract the gene from a normal cell from the mouse's body and
place it within the virus. I will also add an GFP gene, which acts as a
reporter gene.
• 4. I will then place the vector and place it within a platina that has
islet cells (that have been differentiated from stem cells of the same
mouse or from donor mice).
15. Methodology
• 5. The virus will enter the cells by endocytosis.
• 6. Then the virus will deposit its genetic material into the nucleus
where it becomes a molecule of double stranded DNA.
• 7. To make sure the virus deposited its genetic material, I will examine
the cells for the green fluorescent light that the GFP gene gives off.
• 8. Then I will proceed to locate the hepatic portal vein in the mouse
where I will insert the cells.
16. Methodology
• 9. The mouse will receive a local anesthetic and a sedative.
• 10. Then, Diagnostic Radiology will be used to guide the placement of a
thin, flexible tube called a catheter through a small incision in the upper
abdomen and into the greater pancreatic artery.
• 11. The islets are then infused, or pushed, slowly into the pancreas through
the catheter.
• 12. After surgery, It is important that during this time, the islet cells be
given an opportunity to rest in order to fully graft and begin producing
insulin on their own. The mouse will be on a continuous intravenous
infusion of insulin for some time, and its blood glucose levels will be
checked frequently.
17. Future Investigation
• After the recovery time from the surgery, I will inspect to see if the
implanted cells are producing insulin.
• I will monitor Group A and B’s blood sugar by periodically extracting
samples of their blood.
• I will compare to see how these levels vary from the mice with the
CD47 gene to the ones who don’t have this gene.
18. Potential Pitfalls
• The gene could not enter the vector.
• The vector could not infect the islet cells.
• Even though the cell is successfully implanted into the body, the
immune system could over power the immunosuppressive gene.
• The flow of my proposal investigation, was halted by the discovery
that no immunosuppressive gene had been discovered. So the use of
CD47 may not be effective for this treatment.
19. Expected Results
• I would expect to see a greater production of insulin by the Group A
mice than the Group B.
• This would indicate that the introduction of the CD47 gene proved
helpful in the experiment.
20. Why Diabetes?: Facts and Figures
• 387 million people in the world have
diabetes; by 2035 this will rise to
592 million.
• Diabetes is the third leading cause
of death in Puerto Rico and the seventh
leading cause of death in the U.S.
• More than 500,000 of Puerto Rico’s
3.7 million residents suffer
from diabetes.
• In fact, residents of Puerto Rico are 1.8
times as likely to have diagnosed
diabetes as U.S. non-Hispanic whites,
according to CDC statistics.
• Diabetes patients spend an average of
$6,000 annually on costs for treating their
disease.
21. References:
• Wisse, MD, B. (2014). A.D.A.M. Medical Encyclopedia.
• Dansinger, MD, M. (2015, June 22). Type 2 Diabetes: The Basics. WebMD.
• B Online Staff, . (2012, November 16). PR has highest rate of diabetes in US. Caribbean
Business.
• Booth, D.V.M., Ph.D, C. J. (2014, April 10). Diabetes in Rats. AFRMA Rat & Mouse Tales.
Retrieved from http://www.afrma.org/med_diabetes.htm
• Bethesda, MD, . (2009). Stem Cell Basics. In National Institutes of Health. Retrieved from
http://stemcells.nih.gov/info/basics/Pages/Default.aspx
• Konrad, W. (2010, November 12). Protecting Yourself From the Cost of Type 2
Diabetes. The New York Times. Retrieved from
http://www.nytimes.com/2010/11/13/health/13patient.html?_r=1
• Roca, C., Anguela, X., & Ruzo, A. (Writer). (2011). Gene Therapy [Online video].
Barcelona: Universidad Autonoma de Barcelona. Retrieved from
https://www.youtube.com/watch?v=U3RygvuSrok
