gene therapy: the basics - isctmdefinition of gene therapy gene therapy is the introduction of...
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Gene Therapy: The BasicsMark A. Kay MD PhD
Dennis Farrey Family Professor
Stanford University
Definition of gene therapy
Gene therapy is the introduction of nucleic acids (e.g. DNA/genes) into somatic cells of the body to correct or prevent a pathological process
Think of DNA as a class of pharmaceuticals
What do we want to accomplish?
• Add a gene (gene addition) to: supply functionally deficient protein or provide a protein with therapeutic effects (most current clinical trials)
• Fix a mutation in the gene
• Knockdown or knock out a gene: gain-of-function or viral infection
Vectors
Types of vectors used to incorporate exogenous DNA into a cell
Friedman, Scientific American 1997
Vectors used for gene expression strategies must contain a few
essential components
5’ 3’
coding sequenceOr non coding DNA
poly-A site
Other sequences can be included--- Examples: • Enhancers • Introns• Regulatory sequences (to enhance expression or
restrict expression to a specific cell type)
promoter
Potential factors limiting vector efficacy
AAV Vectors
CapRep DNA
AAV
ITR
Therapeutic DNAControlelement
Gene Cassette
DNA
AAV Vectors
ITR ITR
ITR
Pseudotyping Recombinant Vector Genomes
Small number of amino acid changes can have profound effectson the transduction parameters (immunity, efficiency, cell type)
AAV2
AAV1 AAV3 AAV4 AAV5 AAV6
A Problem
Animal Models Do Not Necessarily Predict Human Outcome
Recent Successes
• A form of retinal eye blindness
• Car-T for certain blood cancers
• Immunodeficiency (e.g. Bubble Boy Disease)
• Hemophilias A and B
• Parkinson Disease
Why was hemophilia an early targeted gene therapy
• Small and large animal models that recapitulate the disease
• Well known correlation between level of factor and severity of disease
• As little as a 1 to 2 percent is therapeutic
• No need to have strict gene regulation
Hemophilia B Gene Therapy Trial
What are the challenges?
• How to include patients with pre-existing immunity to the vector
• CTL response and possible insertional mutagenesis
• Getting the dosing right
• How long will it last? Re-administration?
• Manufacturing
What do we want to accomplish?-2Silencing a Gene
Silence a gene
CCR5
Sensitive to HIV
CCR5-Δ32
Resistant to HIV
from a pathogen, gain-of-function mutation
Silencing a Gene by Gene Therapy and RNAi
Genome Editing to Silence a Gene or manipulate specific region of
genomeDifferent nucleases-enzymes that exist in nature that cleave specific DNA sequences. (e.g. CCR5)
These can be modified to recognize a sequence of interest. Examples-homing endonucleases, Zn-finger nucleases, Talens, CRISPR-Cas9
Non-enzyme mediated gene recombination
Sickle Cell Anemia vs Beta- thalassemia
Mysterious RNAs and Gene Therapy
• Non- protein coding RNAs are the dark
matter of the cell
• Over 95% of DNA genome is made into
RNA but only a few percent make proteins
• What do these RNAs do and how can we
manipulate them to treat disease?
The <$1000 Genome
• Neonatal “blood spots” replaced
• Predisposition loci identified
• Health choices and gene therapy prevention (just like a vaccine!!!)
In the not too near future everyone willbe offered genome sequencing
Acceptable Therapy?
• Horrific Diseases to Traits
• Examples: Neuro psychiatric diseases: bipolar, schizophrenia, addiction (OCD), drug reactions, antisocial behavior, intelligence, athletic coordination, sexual preferences etc etc.