Gene therapy for diabetes

1
Gene therapy for diabetes

• Current treatments for diabetics in humans
• Administer externally produced insulin
• Subcutaneous injection
• External pumps
• Nasal sprays
• Problems
• Inconvenience
• Near impossibility of matching insulin dose with
  bodys changing needs
• Alternative treatments
• Transplantation of pancreatic islet cells
• Development of replenishable insulin-secreting
  cell lines for implantation
• gene therapy?

2
Issues to consider

• Need good expression levels
• Need expression levels that respond to the amount
  of sugar in blood
• Need long term expression
• Insulin most be processed by proteolytic enzymes
• Occurs in pancreatic ß cells

Pro-insulin
B
A
C
35 AA
B
C
A
insulin
3
Gene therapy in mice

• Rodent models
• Rats- induced by ß cell toxin
• Autoimmune diabetic mice
• First issue
• How to bypass need for processing
• made a single chain analog (SIA)
• Produced in E. coli
• Shown to have activity- receptor binding, glucose
  uptake ability
• In vivo shown to induce hypoglycemic effect
  (slightly lower than insulin, more than
  proinsulin)

Nature(2000) 408483
B
A
7 AA
B
A
4
Vector design

• pLPK-SIA
• Promoter
• LPK L-type pyruvate kinase
• Activated in response to blood glucose levels
• Albumin leader sequence
• Facilitates secretion from hepatocytes
• SV40 poly-A and enhancer
• downstream of coding sequence to enhance basal
  expression
• Delivery
• Administered plasmid into portal vein of diabetic
  mice
• Detected transfected plasmid only in liver cells

B
A
LPK
SV40
5
Effect of pLPK-SIA

• Looked at toxin-induced diabetic rats
• Injected with pLPK-SIA and pSIA
• Only mice with pLPK-SIA showed any effect
• Blood glucose levels decreased until day 5
• Remained low 4 more days then increased again by
  14 days
• pSIA injected rats were hyperglycemic

6
Expression levels of plasmid

• Looked for plasmid in various tissues 5 days
  after delivery
• Found plasmid sequence in only liver
• Found SIA mRNA only in pLPK-SIA injected rats

7
rAAV-LPK-SIA

• Problem- How to overcome short half-life of
  treatment
• Designed an AAV vector
• Produced rAAV-LPK-SIA virus in 293 cells
• Administered into portal vein of toxin-induced
  diabetic rats
• Looked for effects
• Blood glucose levels decreased to normoglycemic
  levels within 1 week

8
Molecular state of rAAV genome

• AAV is a single stranded DNA genome
• Southern blots of liver genomic DNA from rats
• Probed - SIA cDNA and SV40

9
SIA expression in the liver

• Anti-SIA antibody detects SIA in hepatocytes of
  treated rats
• Plasma SIA levels after glucose administration

Control rats
-Also found that production of SIA was slower
than release of insulin into blood -Insulin is
secreted directly SIA must first be transcribed
10
Will it work in mice?

• Same treatment on mice with autoimmune diabetes
  (NOD)

Plasma SIA levels after glucose loading
Blood glucose levels
Blood glucose levels went to normal after 7 days
and stayed there for 5 months
Levels of SIA in plasma after glucose
administration
11
Genomic integration in mice

• Also determined that no anti-SIA antibodies were
  in circulation, therefore no immune response to
  treatment was elicited

12
Will it work in humans?

• Rodents have different physiology with respect to
  glucose maintenance
• Viral integration in liver cells will lead to
  high exposure of those cells to insulin- may lead
  to hypoglycemia after glucose ingestion
• Biggest obstacle is to have insulin delivered at
  the right dose
• Hypoglycemia can be a problem
• Using LPK promoter is first positive step