Gene Therapy: The Basics - ISCTMDefinition of gene therapy Gene therapy is the introduction of...
Transcript of Gene Therapy: The Basics - ISCTMDefinition of gene therapy Gene therapy is the introduction of...
![Page 1: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/1.jpg)
Gene Therapy: The BasicsMark A. Kay MD PhD
Dennis Farrey Family Professor
Stanford University
![Page 2: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/2.jpg)
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
![Page 3: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/3.jpg)
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
![Page 4: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/4.jpg)
Vectors
Types of vectors used to incorporate exogenous DNA into a cell
Friedman, Scientific American 1997
![Page 5: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/5.jpg)
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
![Page 6: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/6.jpg)
Potential factors limiting vector efficacy
![Page 7: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/7.jpg)
AAV Vectors
CapRep DNA
AAV
ITR
Therapeutic DNAControlelement
Gene Cassette
DNA
AAV Vectors
ITR ITR
ITR
![Page 8: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/8.jpg)
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
![Page 9: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/9.jpg)
A Problem
Animal Models Do Not Necessarily Predict Human Outcome
![Page 10: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/10.jpg)
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
![Page 11: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/11.jpg)
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
![Page 12: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/12.jpg)
Hemophilia B Gene Therapy Trial
![Page 13: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/13.jpg)
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
![Page 14: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/14.jpg)
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
![Page 15: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/15.jpg)
Silencing a Gene by Gene Therapy and RNAi
![Page 16: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/16.jpg)
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
![Page 17: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/17.jpg)
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?
![Page 18: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/18.jpg)
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
![Page 19: Gene Therapy: The Basics - ISCTMDefinition 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](https://reader034.fdocuments.us/reader034/viewer/2022052611/5f08edbc7e708231d42467c2/html5/thumbnails/19.jpg)
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.