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III. Emerging Clinical Applications of CRISPR/Cas9 as Promising Strategies in Gene Therapy and Disease Correction
Chi-Ping Day, Ph.D.Staff Scientist
Laboratory of Cancer Biology and GeneticsNational Cancer Institute
NIH, Bethesda, MD
“Correcting” Disease by Gene Therapy
How It Works:• Intervening disease by delivery of a genetic material into targeted cells.• Restoring a required gene function that is lacking or insufficient in the cells.• Suppressing a dysfunctional gene.
The Promise: A single approach that can correct any dysfunctional cell.
Major Indications:• Inherited disease• Cancer• Infectious disease
Format:• Ex vivo: Cell therapy• In Vivo: Systemic and local treatment
Technical Aspects of Gene Therapy
Effectors:• Gene expression: DNA vectors, RNA oligos.• Gene knockdown: anti-sense RNA, siRNA, shRNA
Technical Issues:• Delivery efficiency• Targeting specificity• Consistency of expression• Immune response• Side effects from vectors
The Now and Then of Gene TherapyCurrent Status: • CAR T cells are closest to get FDA approved.• p53 gene therapy for lung cancer has been approved in China.• Majority of them, however, are still under investigation.
Major Setback in the Past: • Unsustainable expression of delivered genes in several trials.• Jesse Gelsinger's death in 1999.• SCID patients developed leukemia-like condition after HSC treatment.
Hurdles in Clinical Development of Gene TherapyGeneral issues:• Efficiency of gene delivery• Consistency of expression: position effect• Risk of cell transformation• Immune response against vectors and inserted genes
Ex vivo cell therapy:• Selection of the engineered cells.• Amplification of the engineered cells.• Clonal competition
In vivo treatment:• Stability in vivo• Targeting and toxicity
How CRISPR/Cas9 Can Help?
Specific gene editing:• No issue in consistency of gene expression• Avoid position effect• Reducing the risk of cell transformation • Avoiding or reducing immune response against edited
genes
No requirement of constant expression of effectors:• Reducing the risk of cell transformation • Avoiding potential immune response against Cas9
Remaining and New Issues
• Efficiency and specificity of delivery• Immune response against residual expression of effectors• Cell selection• Off-targeting effect• Risk of DNA damage and enhanced cell aging
Possible Solution
• Efficiency and specificity of delivery• Immune response against residual expression of effectors→ Transient reporter
• Cell selection → Transient drug selection
• Off-targeting effect • Risk of DNA damage and enhanced cell aging → Quality monitoring by sequencing → Examining markers of senescence and differentiation
Clinical Development (1): Chimeric Antigen Receptors (CAR) T Cells
Production and Adoptive Transfer of CAR T Cells
Retroviral vectors
First proposed human test of CRISPR passes initial safety reviewScience. June 25, 2016
• T cells genetically edited by TALEN resulted in the remission of leukemia in a one-year-old patient
• Performing three CRISPR edits on T cells from 18 patients with several types of cancers
• Testing safety rather than efficacy• Transfer by a retroviral vector• Edit 1: inserting CAR targeting NY-ESO-1• Edit 2: removing a immune checkpoint PD-1• UPenn will manufacture the edited cells
Clinical Development (2): Gene Editing at Retina
• Eye is an immune-privileged site• Sub-retinal local injection of DNA vector• Electroporation can be applied to eyes• Viral vectors can also be used• Editas Medicine (Cambridge, MA) has announced plans to use CRISPR to treat an
inherited eye disease in 2017, but RAC has not yet reviewed a proposal from the company.
In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa
Molecular Therapy (2016); 24 3, 556–563.
Clinical Development (3): Elimination of HIV-1 Genomes from Human T-lymphoid Cells by
CRISPR/Cas9 Gene EditingScientific Reports 6, No. 22555 (2016)
• In vitro or ex vivo• Lentiviral vector for gene transfer• Whole-genome sequencing and RT-PCR for monitoring off-targeting and viral gene
expression • Requiring bone marrow transplantation for clinical application
CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape
Cell Reports. 15(3): 481–9, 2016
Clinical Development (4): Therapeutic genome editing by combined viral and non-viral delivery
of CRISPR system components in vivoNature Biotechnology 34, 328–333 (2016)
• Lipid nanoparticle–mediated delivery of Cas9 mRNA • Adeno-associated viruses encoding a sgRNA and a repair template • The delivery vectors were trapped in liver of a mouse model of human
hereditary tyrosinemia via intravenous injection• Treatment generating fumarylacetoacetate hydrolase (Fah)-positive
hepatocytes by correcting the causative Fah-splicing mutation and rescuing disease symptoms
• The efficiency of correction was >6% of hepatocytes after a single application
• Cell therapy as a standard treatment Cancer Neuronal regeneration
• Local treatment for inherited diseases Eye-degenerating diseases Hearing-degenerating diseases
• Rheumatoid diseases?
The (Near) Future