Gene Editing - Challenges and Future of CRISPR in Clinical Development

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Genetic Engineering2Physician Led | Therapeutically Focusedthe process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts)(Hsu et al, 2014).

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.


Zinc-Finger Nuclease (ZFN) 3Physician Led | Therapeutically Focused

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 technology3

Transcription Activator Like Effector Nuclease (TALEN): Application in Duchenne Muscular Dystrophy4Physician Led | Therapeutically Focused


DMD geneXp21DMD 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

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 4

CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats5Physician Led | Therapeutically FocusedFirst described in E. coli and determined to be part of the bacterial innate immune system versus bacteriophagesConsists of short segments of DNA that are palindromes interspaced with spacer DNA The spacer DNA is identical in sequence to viral (bacteriophage) DNAThere are additional CRISPR associated proteins: cas proteins that are typically helicases or nucleases



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. 5

CRISPR BasicsPhysician Led | Therapeutically Focused6Physician Led | Therapeutically Focused6



Bacteria Cell Wall

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.6

cas Protein and crRNA ProducedPhysician Led | Therapeutically Focused7Physician Led | Therapeutically Focused7



cas protein /crRNA complex

Bacteria Cell Wall

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 DNA7

Physician Led | Therapeutically Focused8Physician Led | Therapeutically Focused8



cas Protein

A New Bacteriophage ArrivesBacteria Cell Wall

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


Bacteriophage Denied!!New Spacer DNA Incorporated into Bacterial Genome for Next TimePhysician Led | Therapeutically Focused9Physician Led | Therapeutically Focused9

New SpacerDNA



Bacteria Cell Wall

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.9

Physician Led | Therapeutically Focused10The BreakthroughJinek et al, Science, 2012

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 RNAs 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