Gene Therapy

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• Gene Therapy
• GTTB, NIDCR, NIH, DHHS
• John A. Chiorini

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• Gene Therapy is.
• An experimental process that involves altering
  the genetic composition of cells for the
  treatment or prevention of disease.

Cystic Fibrosis Blood Disorders Muscular Dystrophy
Genetic Diseases
Cancer Cardiovascular Neurological
Disorders Infectious Disease (AIDS)
Acquired Diseases
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Gene Delivery Service
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Factors Required for Successful Gene Therapy
1. Uptake of vector by appropriate cells
2. Vector entry into nucleus
?
receptor
?
Endosome
Nuclear Matrix
MHCII
Nucleus
Golgi
CTL
Cytoplasm
3. Evade Host Defense
Courtesy of Mark Kay
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Current Success

• Immune deficiency (xSCIDs, ADA)
• Hemophilia B

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SCIDs Trialsmake the patient their own donor

• Retroviral vector
• (ex vivo) hematopoietic stem cells
• Cells will be returned in single infusion

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SCID Trial Results

• Patients are able to live normal lives at home
  have normal
• numbers of immune cells
• Patients can respond to several childhood
  immunizations,
• (diphtheria, tetanus,polio)
• Corrected deficiency in patients that had poor
  results with
• traditional bone marrow transplantation.
• No need for periodic infusions of protective
  immunoglobulin.
• Risk of inducing leukemia. Insertion mutagenesis
  as a
• result of the vector (3/30)

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FIX Trialshave the patients produce there own
replacement enzyme

• Dependent Parvovirus (AAV)
• (in vivo) IM or Hepatic artery
• Open-label dose escalation design

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FIX trial results

• Transient low level of expression
• Require less frequent injections
• No inhibitor (antibody formation) to
• FIX
• No evidence of germ line transmission
• in liver trial, immune response to transduced
  
• cells (transient transaminitis)

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Promising advances and positive results in some
recent clinical trials. To move
ahead. -improved vectors. -better
understanding of biological interactions of
vector and host.
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Factors Required for Successful Gene Therapy
1. Uptake of vector by appropriate cells
2. Vector entry into nucleus
?
receptor
?
Endosome
Nuclear Matrix
Nucleus
Cytoplasm
Courtesy of Mark Kay
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Factors Required for Successful Gene Therapy
1. Uptake of vector by appropriate cells
?
receptor
?
Endosome
Nuclear Matrix
Nucleus
Cytoplasm
Courtesy of Mark Kay
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Total 1192
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Total 1192
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Hurdles to Overcome

• Delivery System
• Safe
• Effective
• Well tolerated
• Immune Response
• Antibodies to Vector
• T-cells against Target cell
• Therapeutic protein
• Regulated Expression

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The tools of gene therapy
Non-Biologic
Biologic
viruses
Physical and chemical
Retrovirus
Adenovirus
Liposome
Microinject
Parvovirus
(AAV)
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The tools of gene therapy
Physical and chemical
Biological viral vectors
Retrovirus
Adenovirus
AAV
Liposome
Naked DNA
Courtesy of Matt Weitzman
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Gene therapy could be very different for
different diseases

• Gene transplantation
• (to patient with gene deletion)
• Gene correction
• (To revert specific mutation in the gene of
  interest)
• Gene augmentation
• (to enhance expression of gene of interest)
• Targeted killing of specific cells by introducing
  killer gene
• Gene ablation targeted inhibition of gene
  expression

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Methods of Gene Transfer

• Chemical - CaPO4, lipid
• Physical - microinject, electroporate
• Biological - viral

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Vector Categories
Viral-Based - Disarming naturally existing
viruses to effectively and safely transfer
therapeutic genes into patients cells Non-Viral
- Synthetic nanoparticles consisted of genes
encapsulated in lipids or polymers
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Methods of Gene Transfer

• Chemical/Non-Viral - Synthetic nanoparticles
• consisted of genes encapsulated in lipids or
• polymers
• Physical - microinject, electroporate
• Biological/Viral Disarmed, naturally existing
• viruses

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Factors Required for Successful Gene Therapy
1. Uptake of vector by appropriate cells
2. Vector entry into nucleus
?
receptor
?
Endosome
Nuclear Matrix
MHCII
Nucleus
Golgi
CTL
Cytoplasm
3. Evade Host Defense
Courtesy of Mark Kay
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Factors Required for Successful Gene Therapy
1. Uptake of vector by appropriate cells
2. Vector entry into nucleus
?
receptor
?
Endosome
Nuclear Matrix
MHCII
Nucleus
Golgi
CTL
Cytoplasm
3. Evade Host Defense
Courtesy of Mark Kay
30
Factors Required for Successful Gene Therapy
1. Uptake of vector by appropriate cells
2. Vector entry into nucleus
?
receptor
?
Endosome
Nuclear Matrix
MHCII
Nucleus
Golgi
CTL
Cytoplasm
3. Evade Host Defense
Courtesy of Mark Kay
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Genetic code
COOH
OCOH3
COO
Acetylsalicylic acid