Gene Therapy: The Basics Mark 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 promoter coding sequence poly-a site Or non coding DNA 5 3 Other sequences can be included--- Examples: Enhancers Introns Regulatory sequences (to enhance expression or restrict expression to a specific cell type)
Potential factors limiting vector efficacy
AAV Vectors AAV ITR Rep Cap ITR DNA AAV Vectors ITR Control element Gene Cassette Therapeutic DNA ITR DNA
Pseudotyping Recombinant Vector Genomes AAV2 AAV1 AAV3 AAV4 AAV5 AAV6 Small number of amino acid changes can have profound effects on the transduction parameters (immunity, efficiency, cell type)
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?-2 Silencing a Gene Silence a gene from a pathogen, gain-of-function mutation CCR5 Sensitive to HIV CCR5-Δ32 Resistant to HIV
Silencing a Gene by Gene Therapy and RNAi
Genome Editing to Silence a Gene or manipulate specific region of genome Different 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 In the not too near future everyone will be offered genome sequencing Neonatal blood spots replaced Predisposition loci identified Health choices and gene therapy prevention (just like a vaccine!!!)
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.