The Use of Gene Therapy To Treat Type 1 Diabetes Mellitus

Laura V. Díaz-MéndezRISE Program

Prof. Enrique Rodriguez-Borrero, PhD

Research Proposal

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

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.

Insulin Mechanism

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.

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.

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.

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.

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

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.

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.

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

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.

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).

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.

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.

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.

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.

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.

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 theirdisease.

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

Thank you for your attention