Gene Editing - Challenges and Future of CRISPR in Clinical Development
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In this presentation, Medpace experts discuss the unique opportunity of evolving gene editing technology as it applies to human diseases
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Gene Editing - Challenges and Future of CRISPR in Clinical Development
- 1. CLICK TO EDIT TITLE Click to edit subtitle
- 2. Genetic Engineering 2 Physician Led | Therapeutically Focused “…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).
- 3. Zinc-Finger Nuclease (ZFN) 3 Physician Led | Therapeutically Focused
- 4. Transcription Activator Like Effector Nuclease (TALEN): Application in Duchenne Muscular Dystrophy 4 Physician Led | Therapeutically Focused X Y DMD gene Xp21 • 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. CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats 5 Physician Led | Therapeutically Focused 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 Spacer DNA cas Spacer DNA Spacer DNA Spacer DNA
- 6. CRISPR Basics Physician Led | Therapeutically Focused6 Physician Led | Therapeutically Focused6 Spacer DNA cas Spacer DNA Spacer DNA Spacer DNA Bacteria Cell Wall
- 7. cas Protein and crRNA Produced Physician Led | Therapeutically Focused7 Physician Led | Therapeutically Focused7 Spacer DNA cas Spacer DNA Spacer DNA Spacer DNA cas protein /crRNA complex Bacteria Cell Wall
- 8. Physician Led | Therapeutically Focused8 Physician Led | Therapeutically Focused8 Spacer DNA cas Spacer DNA Spacer DNA Spacer DNA cas Protein A New Bacteriophage Arrives Bacteria Cell Wall
- 9. Bacteriophage Denied!! New Spacer DNA Incorporated into Bacterial Genome for Next Time… Physician Led | Therapeutically Focused9 Physician Led | Therapeutically Focused9 New Spacer DNA Spacer DNA cas Spacer DNA Spacer DNA Spacer DNA Bacteria Cell Wall
- 10. Physician Led | Therapeutically Focused10 The Breakthrough Jinek et al, Science, 2012
- 11. 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
- 12. Clinical Human Applications of CRISPR 12 Physician Led | Therapeutically Focused 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. Clinical Human Applications of CRISPR? 13 Physician Led | Therapeutically Focused 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. Human Experience 14 Physician Led | Therapeutically Focused o 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
- 15. Cancer Studies 15 Physician Led | Therapeutically Focused Source: Clinicaltrials.gov, Sept 2016
- 16. The NIH Recombinant Advisory Committee has Approved the First US Trial 16 Physician Led | Therapeutically Focused The University of Pennsylvania: combination of PD-1 and NY-ESO-1 and LAGE-1 in human cancer Time, 2016
- 17. Limitless Applications… Physician Led | Therapeutically Focused17 • Drug development – optimize biotech manufacture • Disease models • Ecological vector control – mosquito sterilization • Biofuels • Agriculture – modification of crop strains or animals
- 19. Clinical Development Considerations for Gene Editing Technology 19 Physician Led | Therapeutically Focused o 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
- 20. Physician Led | Therapeutically Focused 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 20
- 21. 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 Focused21
- 22. 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 Focused22
- 23. 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 Focused23
- 24. Physician Led | Therapeutically Focused 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 Administration 24 o 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. 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 25 Physician Led | Therapeutically Focused
- 26. 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 Focused26
- 27. 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 Physician Led | Therapeutically Focused27 o Health Technology Assessment bodies (HTAs) may need to consider new approaches to pricing Strimvelis (GSK) - pay for performance agreement in Italy (AIFA)
- 28. 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 Focused28
- 29. 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)
- 30. CLICK TO EDIT TITLE Click to edit subtitle Thank you
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- Good morning. Trevor and I would like to thank the audience for their attendance today. Together, Trevor and I will review the basics of gene editing, recent clinical experience with the technology followed by a discussion of the regulatory and ethical implications of human genetic engineering. We will have time for questions at the end. Gene engineering has evolved over time from a method to generate genetic knock-in and knock-out animals to genetic surgery for human diseases The quest for editing the human genome to treat disease has been an ongoing objective of human medicine for many years. The ability to form a precise DNA break, followed by editing or correction has been attempted via a variety of techniques: meganucleases, oligonucleotides, peptide nucleic acids and more recently zinc finger nucleases and transcription activator-like effector nucleases (TALEN). The most recent addition, clustered regularly interspaced short palindromic repeats (CRISPR) has created an increasing level of interest and scrutiny. Like any advance in medicine, genetic engineering offers great promise and great responsibility.
- I will focus the discussion today on three of the gene editing technologies: Zinc finger nuclease, TALEN and CRISPR. In brief, zinc finger nucleases a class of engineered DNA-binding proteins that enable targeted double-strand breaks in DNA at user-selected locations. Each Zinc Finger Nuclease is comprised of a DNA binding domain and a DNA cleaving domain comprised of the nuclease domain of Fok I. When the DNA-binding and DNA-cleaving domains are fused together, a highly-specific pair of 'genomic scissors' are created. Zinc finger nucleases can recognize independently 3-4 DNA bases and when linked can target specific DNA sequences, bind and cleave and either through non homologous end joining or homologous recombination can either replace or mutate the target gene This technology has been used in plant and mammalian cells. The NEJM article here provides an example of a human clinical trial that used ZFN. Following a clinical observation of a less aggressive clinical course in select HIV infected patients who were heterozygous for the CCR5 delta 32 gene combined with the single report of undetectable HIV following an allogeneic cell transplant from a homozygous CCR5 delta 32 donor, a clinical trial was undertaken. the question was raised if you could infuse CD4 T cells that had undergone gene editing rendering them CCR5 delta 32 deficient. 12 patients enrolled in a phase 1 trial involving CCR5 modified CD4 T cells. One of four evaluable patients had undetectable HIV and the trial was felt to be safe with one related SAE. Concerns regarding off target cleavage have been raised with this technology
- A second gene editing technology has been used in clinical trials, TALEN. TALEN: are engineered restriction enzymes that cut specific sequences of DNA. The basic construct consists of a transcription activator-like effectors (TALEs) that is bound to practically any desired DNA sequence, so when combined with a nuclease, a resulting specific DNA cut will occur. Transcription activator-like effector nucleases (TALEN) were the next evolution of chimeric nucleases that are more readily engineered to specific binding domains providing more specificity. Based upon the discovery by Scholze and Boch and colleagues of plant pathogens, TALE nucleases represent a method to target endogenous genes in cells. Clinical applications of TALEN have been published. In 2015, Li and colleagues published on TALEN correction in stem cell correction of the Duchenne muscular dystrophy gene. The DMD gene comprised on 79 exons, deletions and duplications that can lead to the lack of function dystrophin protein. The large size makes traditional adenovirus gene therapy difficult. In the publication, they demonstrated proof of principle. TALEN technology was also reported in the UK with the use of TALEN in chimeric antigen receptor allogeneic modified T cells in pediatric pre B cell ALL where the TCR was edited out using TALEN technology. The limitations of TALEN however are the off target effect
- Now moving on to the next technology: Clustered regularly interspaced short palindromic repeats or CRISPR CRISPR has been known by bacteriologists for many years and first described as an innate immune system used by bacteria to fend off viral infection via bacteriophages. It was noted that there were short segments of palindromic DNA interspaced with spacer DNA. The spacer DNA being unique and has been found to be identical to viral DNA. In addition to the DNA sequence, cas or CRISPR associated genes that encode for proteins are found that are typically helicases or nucleases. As CRISPR is a newer technology, we will take a moment to describe how it functions in bacterial cells.
- Lets go through a very basic description of CRISPR in bacteria. A bacteria is infected by a bacteriophage, the virus injects viral DNA into the bacteria.
- If the bacteria has seen this viral DNA before, the cas protein is transcribed along with transcription crRNA which fits into the cas protein complex and using the helicases and nucleases of the cas protein complex to break apart the viral DNA
- What if there is a novel viral DNA introduced, the CRISPR system will generate a new class 1 cas protein will break apart the viral DNA
- And incorporate the new spacer DNA into the bacterial genome. When infected by the bacteriophage in the future, the bacteria would have adaptive immunity to that virus and use the CRISPR cas system to degrade the DNA. This system was known for some time as an immune system of bacteria. The innovation occurred when several investigators evaluated one CRISPR system in Strep pyogenes.
- In 2012, using Strep pyogenes, Doudna, Charpentier and others described a modified mechanism to use the CRISPR system with the strep pyogenes cas proteins: cas 9. The paper published in Science in 2012, was a revolutionary change in DNA editing as it provided an elegant system that could be used to precisely edit DNA. In the native system, cas9 is a nuclease. There are two RNA’s formed: the crRNA and an additional RNA, the tracrRNA which holds the crRNA in place. The advance described in the paper was the creation of a chimera of the entire system that combined the tracrRNA and crRNA into one guide RNA: the gRNA. Thus the system created is the cas9 protein and the gRNA, the chimera. The system would work very similarly to just described in the simple diagrams in preceding slides. It works by taking the sequence of DNA that you want to edit and creating a gRNA with that exact sequence in the gRNA. Then insert the chimera into the target cell, the cas9 will cut the DNA at the exact sequence. Then cell will direct endogenous repair mechanisms within the cell to repair the cut the DNA either without target sequence of DNA Alternatively, you can insert a gene, in this instance you have the cas9, the gRNA and the host RNA. The system will cut the DNA at the target based on the gRNA, then the host RNA will be inserted into the DNA as a new segment. What was striking about this advance was the elegant simplicity of the method and the explosion of applications that commenced following the publication was astounding.
- With these observations, a flurry of CRISPR activity has commenced. In Dominguez Nature Reviews, there are elegant descriptions of mechanisms to interfere with transcription of a DNA which one could consider in an overexpression state, for example p53. There are applications of co-activation and interference and finally transcription activation In addition to these direct DNA applications, scientists are investigating targeting the epigenome using the CRISPR system.
- Certainly, we can all contemplate the myriad of applications that gene editing could be considered. We have discussed the ZFN approach to HIV infection and Kang and colleagues reported in April of this year with the use of the CRISPR cas 9 system to insert the HIV resistance gene CCR5 into human embryos. The TALEN approach to T cell receptors which one could surmise would be possible fields of clinical investigation.
- Additionally, as we described TALEN technology application in Duchennes, one could consider removal or addition of genetic material as it applies to genetic diseases. Finally, the field of human organ or stem cell transplantation in theory could be a possible application of CRISPR/cas even to the point of using mismatched human or non-human donors which would revolutionize human transplantation.
- Clinical reports are beginning to be published using the CRISPR cas 9 system. In 2015, Liang and colleagues reported on human zygotes where the human beta globin gene was cleaved with the system. They demonstrated low efficiency and off target cleavage but did demonstrate proof of principle. Additional human trials are forthcoming with several companies and academic centers developing CRISPR based systems for human diseases.
- A quick check of the clinicaltrials.gov web page found 4 clinical trials in China that as of Sept 2016 were not recruiting patients but were targeting a variety of human malignancies. As a clinical research organization, it is astounding the speed at which the CRISPR/cas system has gone from bench to bedside and the global reach of this technology.
- And in the US, the University of Pennsylvania received RAC approval for the first US trial using CRISPR/cas technology targeting a combination of PD-1, NY-ESO1 and LAGE-1 in human malignancy.
- Gene editing technology has limitless applications, from drug development, disease models, ecological vector control, biofuels and most recently reported in Sweden modification of crops, in this case a CRISPR modified cabbage and most recently a CRISPR modified cucumber was generated and eaten this month. In this quick review of gene editing technology, I have hopefully touched on a variety of gene editing technologies, given a simple explanation of the newest addition, CRISPR and would like to share the screen with my colleague Trevor Walker who will discuss the regulatory and ethical implications of these technologies.
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