1. 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 Genetics
National Cancer Institute
NIH, Bethesda, MD

2. “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

3. 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

4. The Now and Then of Gene Therapy
Current 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.

5. Hurdles in Clinical Development of Gene Therapy
General 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

6. 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

7. 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

8. 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

9. Clinical Development (1):
Chimeric Antigen Receptors (CAR) T Cells

10. Production and Adoptive Transfer of CAR T Cells
Retroviral vectors

11. First proposed human test of CRISPR passes initial safety review
Science. 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

12. 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.

13. 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.

14. Clinical Development (3):
Elimination of HIV-1 Genomes from Human T-lymphoid Cells by
CRISPR/Cas9 Gene Editing
Scientific 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

15. CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication
and Accelerate Viral Escape
Cell Reports. 15(3): 481–9, 2016

16. Clinical Development (4):
Therapeutic genome editing by combined viral and non-viral delivery
of CRISPR system components in vivo
Nature 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

17. • 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