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