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2 Physician Led | Therapeutically Focused
Genetic Engineering
“…the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts)”
(Hsu et al, 2014).
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Zinc-Finger Nuclease (ZFN)
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Transcription Activator Like Effector Nuclease (TALEN): Application in Duchenne Muscular Dystrophy
X Y
DMD geneXp21 • DMD gene: 79 exons, deletions,
duplications or loss can lead to lack of functional dystrophin protein
• Large size renders traditional AAV based gene editing difficult
• Li et al. (2015) used TALEN to correct in iPSCs via exon knockin and demonstrated proof of principle
• TALEN can have off target mutagenesis
Li et al, Stem Cell 2015
5 Physician Led | Therapeutically Focused
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
o First described in E. coli and determined to be part of the bacterial innate immune system versus bacteriophages
o Consists of short segments of DNA that are palindromes interspaced with spacer DNA
o The spacer DNA is identical in sequence to viral (bacteriophage) DNA
o There are additional CRISPR associated proteins: cas proteins that are typically helicases or nucleases
SpacerDNA
cas SpacerDNA
SpacerDNA
SpacerDNA
Physician Led | Therapeutically Focused6
CRISPR Basics
Physician Led | Therapeutically Focused6
SpacerDNA
cas SpacerDNA
SpacerDNA
SpacerDNA
Bacteria Cell Wall
Physician Led | Therapeutically Focused7
cas Protein and crRNA Produced
Physician Led | Therapeutically Focused7
SpacerDNA
cas SpacerDNA
SpacerDNA
SpacerDNA
cas protein /crRNA complex
Bacteria Cell Wall
Physician Led | Therapeutically Focused8 Physician Led | Therapeutically Focused8
SpacerDNA
cas SpacerDNA
SpacerDNA
SpacerDNA
cas Protein
A New Bacteriophage Arrives
Bacteria Cell Wall
Physician Led | Therapeutically Focused9
Bacteriophage Denied!!New Spacer DNA Incorporated into Bacterial Genome for Next Time…
Physician Led | Therapeutically Focused9
New SpacerDNA
SpacerDNA
cas SpacerDNA
SpacerDNA
SpacerDNA
Bacteria Cell Wall
Physician Led | Therapeutically Focused10
The Breakthrough
Jinek et al, Science, 2012
Physician Led | Therapeutically Focused11
Next?
Physician Led | Therapeutically Focused
o Transcription interference
o Co-activation and interference
o Transcription activation
o Epigenetic modifiers
11
Dominguez et al., Nature Reviews: Molecular Cell Biology, 17, 5-15, Jan 2016
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Clinical Human Applications of CRISPR
o Viral infections: HIV, HSV, VZV Inserted viral genome could be removed by
altering immune cells Human embryos: Kang and colleagues inserted
the CCR5Δ32 allele into early human 3PN embryos
o Cancer Mutation driven cancers
Kang, X. et al. J Assist Reprod Genet 33, 581, 2016
13 Physician Led | Therapeutically Focused
Clinical Human Applications of CRISPR?
o Genetic diseases Remove or add the sequence
that is causing the disease
o Transplantation Gene editing of mismatched human
or even non-human mammals as potential organ donors
Editing will reduce risk of immune responses and rejection when using mismatched organs/tissues/cells
14 Physician Led | Therapeutically Focused
Human Experienceo Liang et al using human tripronuclear
zygotes cleaved the HBB gene with a CRISPR/Cas9-mediated system Low efficiency and edited embryos
were mosaic with off target cleavage
o Other clinical trials forthcoming: Editas CRISPR Therapeutics Caribou Biosciences Intellia Therapeutics
Liang et al, Protein and Cell 2015
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Cancer Studies
Source: Clinicaltrials.gov, Sept 2016
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The NIH Recombinant Advisory Committee has Approved the First US Trial
The University of Pennsylvania:combination of PD-1 and NY-ESO-1 and LAGE-1in human cancer
Time, 2016
Physician Led | Therapeutically Focused17
Limitless Applications…
• Drug development – optimize biotech manufacture
• Disease models• Ecological vector control –
mosquito sterilization• Biofuels• Agriculture – modification of
crop strains or animals
Regulatory and Ethical
Implications
19 Physician Led | Therapeutically Focused
Clinical Development Considerations for Gene Editing Technologyo Therapies may provide life-long cure through a single
treatment
o CRISPR technology has made gene-editing much more accessible and has broadened the range of targets
o Regulatory and ethical frameworks
o Bring new therapies to the clinic via a safe and rapid pathway
Physician Led | Therapeutically Focused20
o Most likely to be largest area of clinical development using CRISPR technology
o Regulations available to govern these applications
o No new ethical concerns/issues
o Research permitted
o Therapies being developed
o Considered for some indications – would alter the genome in all cells and become heritable
o Ethical concerns
o Inconsistent and variable guidelines and regulation
o Limited research permitted
o Development of therapies may be restricted
Somatic Cell Therapy Germ Line Therapy
Physician Led | Therapeutically Focused21
Ethical Concerns
o International Summit on Human Gene Editing (Dec 2015) Concerns over germ line editing – need for an ongoing forum
o NAS/NAM Meeting (April 2016) All aspects of human germline editing, consequences, regulation and potential
applications Committee assembled to perform a year-long in-depth study
o EU CT Directive (2001/20/EC) does not allow germ line editing (Article 9)
o NIH RAC will not review proposals on germ line editing
o Eugenic practices prohibited: Oviedo Convention Convention on Human Rights and Biomedicine (1997) Article 3(2) of the Charter of Fundamental Rights of EU prohibits eugenic practices
Non-Therapeutic Use / Enhancement
Physician Led | Therapeutically Focused22
Regulatory Challenges
o CRISPR/Cas-based gene editing of somatic cell therapies will use in vivo or ex vivo strategies
o Current regulations for gene therapy and cell therapy will regulate CRISPR-based therapies
o Regulators will need to stay up to date with rapid technology advances
o Pathways to market will need to be flexible and allow timely patient access to therapies
Somatic Cell Therapies
Physician Led | Therapeutically Focused23
o Off-target effects/genotoxicity Improvements in targeting of CRISPR/Cas9 system
• Methods to assess genome-wide off-target effects• Need to ensure there is no detectable germ line modification
o Efficacy Single administration may be sufficient but need to consider multiple
treatments Control of CRISPR/Cas editing
o Animal models Significant area for regulators and companies
Safety and Efficacy
Physician Led | Therapeutically Focused24
o Complex manufacturing processes
o Release testing
o Shelf-life may be short (hours)
o Stability and transportation logistics are important considerations
Quality/Manufacturing and Administrationo Patients may be treated in
specialized centers
o Manufacture based at site of administration
o Patients to be localized at these sites – international travel for treatment will become more common
25 Physician Led | Therapeutically Focused
o Role of RAC in the US Recent revisions to streamline the process
o Review of gene therapy studies in EU Additional time for review, may involve expert committees Use of modified viral vectors requires authorisation for use of GMOs
o Regulators gain experience with gene editing therapies (quality, safety, efficacy) Increased focus on review by ECs/IRBs may raise additional
questions and impact the start-up process for CTs
Clinical Trial Considerations
Physician Led | Therapeutically Focused26
Regulatory Pathways to the Market
o EU, Japan and US recognize the importance of faster transit through the current regulatory pathways
EU – Adaptive pathways (conditional approvals), PRIME (PRIority MEdicines), accelerated assessment
Japan –PMD Act, expedited approval system for regenerative medicine products
US – fast track, breakthrough therapy, accelerated approval, priority review
Timely and Flexible; Faster Access to Developing Therapies
Physician Led | Therapeutically Focused27
Cost and Reimbursement
o CRISPR-based therapy that provides a one-off lifetime cure will come at a high development cost Rare diseases will only have a small number
of potential patients
o Single high-cost therapy treatment could remove a lifetime’s cost of existing treatment and be more effective
The Market Challenge
o Health Technology Assessment bodies (HTAs) may need to consider new approaches to pricing Strimvelis (GSK) - pay for performance agreement in Italy (AIFA)
Physician Led | Therapeutically Focused28
Future Direction of Clinical Development
o CRISPR/Cas9-based gene editing of cells and tissues will be an exciting and rapid area of development in the coming years Anticipate that rare genetic diseases, mutation-driven malignancies
and cardiovascular indications will be key areas of development Recognize that the potential is vast and applications may be limitless
o Flexible approach to the regulatory pathways is essential Developers, ethicists and regulators should be discussing
collaboratively at an early stage and throughout the development pathway
o Long term efficacy and safety will demonstrate the value of this technology and demonstrate its potential to treat complex and challenging diseases
Physician Led | Therapeutically Focused29
“Genome editing holds great promise to provide a precise set of tools for counteracting genetic
diseases.
But as Spiderman cautions, ‘With great power, there must come great responsibility’
Moving these methods to clinical applications must proceed judiciously….(and) under appropriate
regulatory oversight”
Kohn et al, Blood (2016, 127: 2553 - 2560)
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