10. Genome editing tools are sequence-specific nucleases
van der Oost. Science (2013) 339:768.
Genome editing tools have
two features:
1) Recognize specific DNA
sequences (i.e. specific
genes or non-coding
elements)
11. Genome editing tools are sequence-specific nucleases
van der Oost. Science (2013) 339:768.
Genome editing tools have
two features:
1) Recognize specific DNA
sequences (i.e. specific
genes or non-coding
elements)
12. Genome editing tools are sequence-specific nucleases
van der Oost. Science (2013) 339:768.
Genome editing tools have
two features:
1) Recognize specific DNA
sequences (i.e. specific
genes or non-coding
elements)
13. Genome editing tools are sequence-specific nucleases
van der Oost. Science (2013) 339:768.
Genome editing tools have
two features:
1)Recognize specific DNA
sequences (i.e. specific
genes or non-coding
elements)
2)Cut DNA (“nuclease”),
then a scar is left behind
14. Genome editing: cleavage repair can either disrupt original
sequence or replace it with a new copy
NEB.com
15. Genome editing: cleavage repair can either disrupt original
sequence or replace it with a new copy
NEB.com
“delete”
16. Genome editing: cleavage repair can either disrupt original
sequence or replace it with a new copy
NEB.com
“delete” “copy and paste”
17. Two strategies for genetic therapy:
gene addition and genome editing
Fischer. Nature (2014) 510:226.
18. Two strategies for genetic therapy: addition and editing
• Gene addition:
• Feasible with existing technology; clinical trials ongoing.
• Early trial results appear exciting.
• Challenges:
1. Will enough of the added gene be made in the cells with the integration?
Will enough of the blood stem cells have the added gene?
2. Is the benefit durable? Will the added gene continue to function over days,
weeks, months, years, decades?
3. Is the added gene safe? Will its semi-random integration into the genome
change the function of other genes in the genome?
Fischer. Nature (2014) 510:226.
19. Two strategies for genetic therapy: addition and editing
• Gene editing:
• Promise of permanent repair of the underlying disease-causing mutation.
• Promise of specific beneficial change at the intended genomic site (e.g. b-
globin gene) without impacting remainder of genome.
• Challenges:
1. Technology is in a relatively early stage and needs to be further
developed.
2. Can enough cells be edited to have therapeutic impact?
3. Will the editing be exquisitely specific, or will other regions of the genome
aside from the target be affected?
Fischer. Nature (2014) 510:226.
27. • Collect blood stem cells from patient with b-thalassemia
• Introduce sequence-specific nucleases to disrupt BCL11A enhancer
• Reinfuse modified blood stem cells to patient
The vision: mimicking common protective genetic variation for
therapeutic benefit
28. • Collect blood stem cells from patient with b-thalassemia
• Introduce sequence-specific nucleases to disrupt BCL11A enhancer
• Reinfuse modified blood stem cells to patient
Potential benefits:
• Loss of BCL11A expression in red blood cells causing increased fetal hemoglobin
• Spares BCL11A expression in other blood cells
• Modification would be permanent
• Survival advantage of cells (would outcompete unmodified cells)
• Compared to gene addition, no semi-random insertion of material into the genome,
and no need for lifelong expression of foreign sequences
The vision: mimicking common protective genetic variation for
therapeutic benefit
29. • Collect blood stem cells from patient with b-thalassemia
• Introduce sequence-specific nucleases to disrupt BCL11A enhancer
• Reinfuse modified blood stem cells to patient
Potential benefits:
• Loss of BCL11A expression in red blood cells causing increased fetal hemoglobin
• Spares BCL11A expression in other blood cells
• Modification would be permanent
• Survival advantage of cells (would outcompete unmodified cells)
• Compared to gene addition, no semi-random insertion of material into the genome,
and no need for lifelong expression of foreign sequences
Potential risks:
• Genome modification at sites other than the intended target
• Preparation (“conditioning”) therapy for stem cell transplant (shared risk of both
gene addition and genome editing; potentially much less toxic than for
“allotransplant” (from related or unrelated donor)
The vision: mimicking common protective genetic variation for
therapeutic benefit
30. Summary
• b-thalassemia results from mutations in b-globin, a
single gene within a large genome
• Gene addition adds a copy of b-globin by semi-
random integration into the genome
– Currently being tested in early-phase clinical trials
– Challenges include: durable high-level expression; ensuring
other important genes are not disrupted due to integration
• Genome editing offers the promise of precise and
permanent genome modification to mimic protective
genetic variation (e.g. at BCL11A) or to repair b-globin
– Challenges include: effective delivery of genome editing tools
to cells to achieve efficient target disruption/repair; ensuring
modification is limited to intended target
31. Acknowledgments
Boston Children’s
Ellis Neufeld
David Williams
David Nathan
Jennifer Eile
Alan Cantor
Bill Pu
Dana-Farber Cancer Institute
GC Yuan
Luca Pinello
Broad Institute
Feng Zhang
Neville Sanjana
Ophir Shalem
Boston Children’s Hospital
Stuart Orkin
Jian Xu
Vijay Sankaran
Sophia Kamran
Matthew Canver
Carrie Lin
Abhishek Dass
Yuko Fujiwara
Zhen Shao
E. Crew Smith
Cong Peng
Hojun Li
Montreal Heart Institute
Guillaume Lettre
Samuel Lessard
Stanford University
Matthew Porteus
Richard Voit
University of Washington
John Stamatoyannopoulos
Peter Sabo
Jeff Vierstra
32. • Feasibility
The vision: mimicking common protective genetic variation for
therapeutic benefit
ASH 2013 Abstracts #434 and 4213. Slide courtesy of Sangamo BioSciences.
33. • Feasibility
The vision: mimicking common protective genetic variation for
therapeutic benefit
Tebas et al. NEJM (2014) 370:901.
Editor's Notes
Thanks so much for this opportunity to present to you today and to participate in this outstanding conference. I want to emphasize to you that this presentation is meant as a vision of future opportunities and as a window into exciting scientific discoveries. Genome editing for thalassemia is still at the preclinical research stage and not yet ready for clinical trials.
I wanted to start with a brief refresher on genetics. The cell is the basic subunit of the human body. Within each cell is a compartment called the nucleus that contains the entire genome, the full set of instructions for making a cell, a tissue, an organ, even a person. The genome is divided into 46 chromosomes, which each contain many genes. The genetic material is composed of the chemical DNA.
The precise sequence of DNA, more than 3 billion individual nucleotides, carries the information of the genome.
All this DNA is tightly packaged into the nucleus, somewhat analogous to a ball of thread.
The genome includes about 20,000 genes.
Most of the genome is not actually the genes but the intervening DNA, the so-called non-coding DNA, which includes elements which control how the genes are turned on and off.
Genome editing offers the promise to perform a sort of genetic surgery, to go into the genome of 3 billion positions and to cut out just one position causing a disease.
Another opportunity for genome editing would be to remove a disease-causing gene and replace it with a healthy gene.
Genome editing takes advantage of tools called sequence-specific nucleases. These tools have two features. First they are able to recognize a specific DNA sequence. This function is depicted in blue.
So within this large genome packaged into the nucleus, these tools can seek out a single gene at a time.
Here they are depicted as this red star, binding at one spot and nowhere else in the genome.
The second step is after they recognize specific DNA sequences then they cut. That is what is indicated here in yellow. Just like when we cut our skin, our body can heal, although it may leave behind a scar. After the genome is cut, the cell is able to repair the cut but a scar may be left behind. This scar is what allows permanent modification of the genome.
This modification can take two forms. Here is depicted the CRISPR/Cas9 genome editing tool. On the left, after cleavage the genome is repaired with small insertion or deletion mutations.
This is analogous to using the “delete” function on a word processor.
On the right, the genome can repair by using a similar copy of the recognized gene or element to precisely swap in new sequence. In the case of a disease-causing mutation, a healthy copy of the gene could be swapped in. This is analogous to the “copy and paste” function of the word processor.
So now there are two strategies for gene therapy for genetic diseases. The first is gene addition as mentioned by Dr Thompson. Let’s say that the cells of the body have the disease causing mutation depicted as a red circle. After the new gene is added the mutant gene is still present. The second strategy is genome editing. In this approach, the target site, in this case the disease-causing red circle mutation, is permanently modified.
Gene addition relies on the semi-random integration of the added gene into the genome.
In contrast genome editing allows the direct and permanent modification of a single gene in the genome.
Thalassemia is caused by mutations of the alpha or beta globin genes. Adult hemoglobin is composed of two alpha and two beta chains, so mutation of either alpha or beta globin disrupts production of adult hemoglobin.
I’m going to focus specifically on beta thalassemia. Many different mutations, as indicated by these triangle, diamond, and arrow symbols can cause beta thalassemia. These are distributed all over the beta-globin gene. There are even more mutations not listed here.
The problem in beta thalassemia is an imbalance in the ratio of alpha to beta globin. There is too much alpha globin relative to beta globin, which results in all of the clinical problems.
Beta-globin is expressed from a cluster of similar beta-like globin genes that are subject to developmental regulation. Gamma-globin, the constituent of fetal hemoglobin, is highly expressed during fetal life. After birth, following the so-called hemoglobin switch, beta-globin is highly expressed but there remains low-level production of residual gamma-globin and fetal hemoglobin. This is the basis by which babies with beta thalassemia are born healthy. Beta thalassemia only develops during the first or second year of life once fetal hemoglobin levels wane. Rare individuals who have a condition called HPFH who completely lack beta globin genes do not have the clinical problems of beta thalassemia because fetal hemoglobin is able to fully compensate.
Mention natural history.
Here I am summarizing a lot of recent scientific progress which has taught us a major mechanism of how fetal hemoglobin is turned off after birth. There is a gene called BCL11A that represses or turns off fetal hemoglobin. Therefore problems of adult hemoglobin such as beta thalassemia, or as is depicted here sickle cell disease, are able to manifest as serious disease.
We recently discovered that some people have natural variation at BCL11A, not in the gene itself, but in an adjacent element which turns on BCL11A called an enhancer. These mutations or polymorphisms are actually helpful. In healthy people these result in modest elevation of fetal hemoglobin levels, which has no consequence. But in individuals with beta-globin disease like thalassemia these variants result in a somewhat milder severity of disease, due to less BCL11A and thus more fetal hemoglobin.
In laboratory experiments we were able to show that complete disruption of this enhancer can result in complete absence of BCL11A, dramatic increase in fetal hemoglobin level, and what we speculate could be major improvement in disease.
