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.