The
n e w e ng l a n d j o u r na l
of
m e dic i n e
clinical implications of basic research Elizabeth G. Phimister, Ph.D., Editor
Translating the Genomic Revolution — Targeted Genome Editing in Primates Toni Cathomen, Ph.D., and Stephan Ehl, M.D. The promise of the genomics revolution is appealing, ambitious, and multipronged. Should this promise be delivered on, patients, equipped with interpretations of their genomes, will be able to gauge the risk of receiving a diagnosis of cancer or heart disease. Physicians will be able to quickly pinpoint the genetic basis of a disorder and take appropriate actions to treat the disease using specific “genome editing” tools. Moving the field one step closer to realizing this promise, Niu et al.1 and Liu at al.2 recently reported the use of designer enzymes (called endonucleases) to introduce specific changes into the genome of one-cell monkey embryos. The subsequent birth of transgenic monkeys provided the first proof of principle of targeted gene editing in totipotent stem cells in primates. Targeted genome editing with designer endonucleases has become increasingly popular; it has been recognized as “Method of the Year” or “Breakthrough of the Year” in two publications over the past 3 years.3,4 Endonucleases are enzymes that cut DNA; in so doing, they introduce a double-strand break into the DNA (Fig. 1 and 2). Endonucleases can be engineered so that they cut the DNA at a specific site in the genome of cells in model organisms or humans. The presence of a double-strand break activates cellular DNA-repair mechanisms, which can be harnessed to introduce specific changes in the genome, such as the insertion of targeted mutations to knock out gene function — or, conversely, to effect the restoration of a mutated DNA sequence. Designer endonucleases come in different “flavors.” Meganucleases and zinc-finger nucleases have been used for several years, and two newcomers have been developed in just the past few years: transcription activator–like effector nucleases (TALENs) and RNA-guided nucleases based on the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system. An advantage of the newer nucleases is their 2342
versatility: they can cut just about any desired DNA target sequence. Niu et al.1 and Liu et al.2 microinjected nucleic acids encoding CRISPR–Cas9 or TALEN into one-cell monkey embryos to disrupt the genes Ppar-γ and Rag1 or Mecp2, respectively. The zygotes were then transferred into surrogate females, and babies born after full-term pregnancies carried the mutations in the respective target genes. As observed when similar approaches have been used in other species, the founder animals had complex genotypes, probably because the designer endonuclease–mediated genome editing did not occur in the singlecell zygote but later in embryogenesis. A more thorough characterization of the genome modifications in the different cell types of the animals, as well as the characterization of the phenotypes of these newborn monkeys, remains to be performed. Even so, this is a relevant advance for the generation of primate models: the rapid Figure 1 (facing page). Generation of Gene-Edited Monkeys with the Use of the CRISPR–Cas9 System. Engineered RNA-guided nucleases are based on the natural CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system used by prokaryotes as a defense against invading DNA. They consist of a complex formed by the Cas9 cleavage enzyme and a guide RNA (gRNA) strand that directs the enzyme to a target site of 20 nucleotides (red). Exchanging a specific portion (red) of the gRNA molecule allows researchers to redirect the Cas9 cleavage activity to a user-defined target sequence. The procedure is as follows: the messenger RNA (mRNA) encoding the Cas9 protein and the gRNA are microinjected into fertilized one-cell embryos. After translation of the Cas9 protein, the Cas9–gRNA complex forms and cleaves the target gene. The resulting DNA double-strand break activates cellular DNA-repair mechanisms, such as nonhomologous end-joining, which is harnessed to introduce a targeted mutation in order to knock out a gene function. The genetically modified zygotes are transferred into surrogate mothers, which give birth to the founder animals that have the desired mutation (here RAG1−/−).
n engl j med 370;24 nejm.org june 12, 2014
The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
Clinical Implications of Basic Research
Microinjection of gRNA and Cas9 mRNA into fertilized one-cell embryo
gRNA
Embryo
gRNA binds to Cas9
Cas9
Active site
Cas9–gRNA complex binds to DNA target
DNA
Pipette
Cas9 creates double-strand break
RAG1 gene
Target sequence
Disrupted RAG1 gene Repair processes triggered
Targeted mutation inserted
Birth of founder animals that lack RAG1
Transfer of modified embryos to surrogate mother
n engl j med 370;24
nejm.org
COLOR FIGURE
june 12, 2014
Draft 4
2343
5/22/14
Author Cathomen The New England Journal of Medicine 1 # Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses Fig without permission. Translating the Genomic Revolution: Title Copyright © 2014 Massachusetts Medical Society. All rights reserved. Targeted Genome Editing ME
Sarlo
The
n e w e ng l a n d j o u r na l
Microinjection of TALEN expression plasmids into fertilized one-cell embryo
of
m e dic i n e
Right TALEN
Nuclease (DNA-cutting) domain
Custom-designed TAL effector DNA-binding domain
Embryo
Custom-designed TAL effector DNA-binding domain
Left TALEN Pipette
MECP2 gene Target sequence
TALEN binds to target sequences and cleaves DNA
Cleaved DNA
Target sequence Repair processes triggered
Disrupted MECP2
Targeted mutation inserted
Transfer of modified embryos to surrogate mother
Birth of MECP2 mutant female monkeys
Death of MECP2 mutant male embryos
generation of monkey models is particularly attractive for research on neuropsychiatric disorders, because the mouse brain is a relatively poor physiological model for the human brain. But how can this approach be further developed for the treatment of human disease? 2344
Disrupting a gene is certainly simpler than Draft 5 5/28/14 correcting one or two dysfunctional gene copies, Author Cathomen as would be necessary inFigthe of many auto2 # case Translating the Genomic Revolution: somal recessive diseases.TitleMoreover, the correcTargeted Genome Editing tion of aberrant nucleotides requires a template, ME Sarlo Phimister which poses additional DE biologic and technical Artist N Koscal COLOR FIGURE
AUTHOR PLEASE NOTE:
n engl j med 370;24
nejm.org
june 12, 2014
Figure has been redrawn and type has been reset Please check carefully
Issue date
6/12/2014
The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
Clinical Implications of Basic Research
Figure 2 (facing page). Generation of Gene-Edited Monkeys with the Use of TALEN. The engineered TALE (transcription activator–like effector) DNA-binding domain of TALENs (TALE nucleases) are based on natural TALEs used by plant pathogenic bacteria to promote their replication in host cells. A TALEN monomer consists of a DNA cleavage domain derived from the restriction enzyme FokI fused to the engineered TALE DNA-binding domain that directs the enzyme to the user-defined target subsite (red). The procedure is as follows: expression plasmids encoding either TALEN monomer are microinjected into a fertilized one-cell embryo. On expression of both TALEN monomers, each monomer binds to its respective target subsite, which allows the two nuclease domains to dimerize and cleave the DNA. The resulting cut activates the error-prone nonhomologous end-joining DNA-repair pathway, which leads to the insertion of mutations at the cleavage site. The genetically modified embryos are transferred to a surrogate mother, which gives birth to founder animals that carry the desired knockout mutation.
challenges. Targeted gene inactivation, as applied in the monkeys, could be used to treat disorders caused by heterozygous gain-of-function or dominant-negative mutations. (A dominant-negative mutation results in the interference of the mutant protein with the function of the nonmutant protein.) Diseases of the hematopoietic system, such as primary immunodeficiencies caused by gain-of-function mutations in STAT1, encoding the signal transducer and activator of transcription 1 protein, is an example of such a disorder. Although the study of CRISPR–Cas9 and TALEN is still in its early stages, many major breakthroughs in improving these two nuclease platforms have recently been made, including modifications to improve the specificity of targeting to a specific site in the genome. Off-target cleavage — potentially resulting in mutagenic events that lead to a malignant phenotype — is a major concern, especially if it occurs in multipotent stem cells destined to be transplanted in patients. Hence, the specificity of engineered nucleases will be the key factor in the translation of this new line of gene therapy into clinical practice. The first human application of designer endonucleases was recently described in the Journal.5 Zinc-finger nucleases were used to disrupt the gene encoding the human immunodeficiency virus (HIV) coreceptor CCR5 in autologous CD4 T cells isolated from, and then transfused back into, persons with HIV infec-
tion, with the goal of engineering cellular resistance to HIV. The study showed that infusion of CCR5-modified T cells was safe, although the sample was small (12 patients) and follow-up was limited (36 weeks); further tests of safety involving a larger sample and long-term followup have yet to be reported. To maintain a durable therapeutic effect, it may be necessary for gene editing to be targeted to the DNA of stem cells. Postnatal somatic stem cells, such as hematopoietic stem cells, are the most accessible cell type for targeting, but increasingly successful protocols for differentiating induced pluripotent stem cells give hope that an approach involving the harvesting of somatic cells, induction of pluripotency, and correction of mutation, followed by differentiation and transplantation, will become a reality.6 The successful genetic editing of one-cell primate embryos raises ethical issues that go beyond the balance between the scientific value of transgenic monkey models in understanding human disease and concerns about their creation. These studies bring us one step closer to the potential for manipulating genes in human embryos. With an open attitude and the will to promote discussions of ethics, scientists developing DNA-editing technologies have the opportunity to engage in and enrich public discourse, in addition to working toward the alleviation of human suffering from genetic diseases. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. From the Center for Chronic Immunodeficiency (T.C., S.E.), the Institute for Cell and Gene Therapy (T.C.), and the Department of Pediatrics and Adolescent Medicine (S.E.), University Medical Center Freiburg, Freiburg, Germany. 1. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cy-
nomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014;156:836-43. 2. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell 2014; 14:323-8. 3. de Souza N. Primer: genome editing with engineered nucleases. Nat Methods 2012;9:27. 4. Genetic microsurgery for the masses. Science 2013;342: 1434-5. 5. Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014;370:901-10. 6. Garate Z, Davis BR, Quintana-Bustamante O, Segovia JC. New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells. Hum Gene Ther 2013;24:571-83. DOI: 10.1056/NEJMcibr1403629 Copyright © 2014 Massachusetts Medical Society.
n engl j med 370;24 nejm.org june 12, 2014
The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
2345
n e w e ng l a n d j o u r na l
of
m e dic i n e
clinical implications of basic research Elizabeth G. Phimister, Ph.D., Editor
Translating the Genomic Revolution — Targeted Genome Editing in Primates Toni Cathomen, Ph.D., and Stephan Ehl, M.D. The promise of the genomics revolution is appealing, ambitious, and multipronged. Should this promise be delivered on, patients, equipped with interpretations of their genomes, will be able to gauge the risk of receiving a diagnosis of cancer or heart disease. Physicians will be able to quickly pinpoint the genetic basis of a disorder and take appropriate actions to treat the disease using specific “genome editing” tools. Moving the field one step closer to realizing this promise, Niu et al.1 and Liu at al.2 recently reported the use of designer enzymes (called endonucleases) to introduce specific changes into the genome of one-cell monkey embryos. The subsequent birth of transgenic monkeys provided the first proof of principle of targeted gene editing in totipotent stem cells in primates. Targeted genome editing with designer endonucleases has become increasingly popular; it has been recognized as “Method of the Year” or “Breakthrough of the Year” in two publications over the past 3 years.3,4 Endonucleases are enzymes that cut DNA; in so doing, they introduce a double-strand break into the DNA (Fig. 1 and 2). Endonucleases can be engineered so that they cut the DNA at a specific site in the genome of cells in model organisms or humans. The presence of a double-strand break activates cellular DNA-repair mechanisms, which can be harnessed to introduce specific changes in the genome, such as the insertion of targeted mutations to knock out gene function — or, conversely, to effect the restoration of a mutated DNA sequence. Designer endonucleases come in different “flavors.” Meganucleases and zinc-finger nucleases have been used for several years, and two newcomers have been developed in just the past few years: transcription activator–like effector nucleases (TALENs) and RNA-guided nucleases based on the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system. An advantage of the newer nucleases is their 2342
versatility: they can cut just about any desired DNA target sequence. Niu et al.1 and Liu et al.2 microinjected nucleic acids encoding CRISPR–Cas9 or TALEN into one-cell monkey embryos to disrupt the genes Ppar-γ and Rag1 or Mecp2, respectively. The zygotes were then transferred into surrogate females, and babies born after full-term pregnancies carried the mutations in the respective target genes. As observed when similar approaches have been used in other species, the founder animals had complex genotypes, probably because the designer endonuclease–mediated genome editing did not occur in the singlecell zygote but later in embryogenesis. A more thorough characterization of the genome modifications in the different cell types of the animals, as well as the characterization of the phenotypes of these newborn monkeys, remains to be performed. Even so, this is a relevant advance for the generation of primate models: the rapid Figure 1 (facing page). Generation of Gene-Edited Monkeys with the Use of the CRISPR–Cas9 System. Engineered RNA-guided nucleases are based on the natural CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system used by prokaryotes as a defense against invading DNA. They consist of a complex formed by the Cas9 cleavage enzyme and a guide RNA (gRNA) strand that directs the enzyme to a target site of 20 nucleotides (red). Exchanging a specific portion (red) of the gRNA molecule allows researchers to redirect the Cas9 cleavage activity to a user-defined target sequence. The procedure is as follows: the messenger RNA (mRNA) encoding the Cas9 protein and the gRNA are microinjected into fertilized one-cell embryos. After translation of the Cas9 protein, the Cas9–gRNA complex forms and cleaves the target gene. The resulting DNA double-strand break activates cellular DNA-repair mechanisms, such as nonhomologous end-joining, which is harnessed to introduce a targeted mutation in order to knock out a gene function. The genetically modified zygotes are transferred into surrogate mothers, which give birth to the founder animals that have the desired mutation (here RAG1−/−).
n engl j med 370;24 nejm.org june 12, 2014
The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
Clinical Implications of Basic Research
Microinjection of gRNA and Cas9 mRNA into fertilized one-cell embryo
gRNA
Embryo
gRNA binds to Cas9
Cas9
Active site
Cas9–gRNA complex binds to DNA target
DNA
Pipette
Cas9 creates double-strand break
RAG1 gene
Target sequence
Disrupted RAG1 gene Repair processes triggered
Targeted mutation inserted
Birth of founder animals that lack RAG1
Transfer of modified embryos to surrogate mother
n engl j med 370;24
nejm.org
COLOR FIGURE
june 12, 2014
Draft 4
2343
5/22/14
Author Cathomen The New England Journal of Medicine 1 # Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses Fig without permission. Translating the Genomic Revolution: Title Copyright © 2014 Massachusetts Medical Society. All rights reserved. Targeted Genome Editing ME
Sarlo
The
n e w e ng l a n d j o u r na l
Microinjection of TALEN expression plasmids into fertilized one-cell embryo
of
m e dic i n e
Right TALEN
Nuclease (DNA-cutting) domain
Custom-designed TAL effector DNA-binding domain
Embryo
Custom-designed TAL effector DNA-binding domain
Left TALEN Pipette
MECP2 gene Target sequence
TALEN binds to target sequences and cleaves DNA
Cleaved DNA
Target sequence Repair processes triggered
Disrupted MECP2
Targeted mutation inserted
Transfer of modified embryos to surrogate mother
Birth of MECP2 mutant female monkeys
Death of MECP2 mutant male embryos
generation of monkey models is particularly attractive for research on neuropsychiatric disorders, because the mouse brain is a relatively poor physiological model for the human brain. But how can this approach be further developed for the treatment of human disease? 2344
Disrupting a gene is certainly simpler than Draft 5 5/28/14 correcting one or two dysfunctional gene copies, Author Cathomen as would be necessary inFigthe of many auto2 # case Translating the Genomic Revolution: somal recessive diseases.TitleMoreover, the correcTargeted Genome Editing tion of aberrant nucleotides requires a template, ME Sarlo Phimister which poses additional DE biologic and technical Artist N Koscal COLOR FIGURE
AUTHOR PLEASE NOTE:
n engl j med 370;24
nejm.org
june 12, 2014
Figure has been redrawn and type has been reset Please check carefully
Issue date
6/12/2014
The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
Clinical Implications of Basic Research
Figure 2 (facing page). Generation of Gene-Edited Monkeys with the Use of TALEN. The engineered TALE (transcription activator–like effector) DNA-binding domain of TALENs (TALE nucleases) are based on natural TALEs used by plant pathogenic bacteria to promote their replication in host cells. A TALEN monomer consists of a DNA cleavage domain derived from the restriction enzyme FokI fused to the engineered TALE DNA-binding domain that directs the enzyme to the user-defined target subsite (red). The procedure is as follows: expression plasmids encoding either TALEN monomer are microinjected into a fertilized one-cell embryo. On expression of both TALEN monomers, each monomer binds to its respective target subsite, which allows the two nuclease domains to dimerize and cleave the DNA. The resulting cut activates the error-prone nonhomologous end-joining DNA-repair pathway, which leads to the insertion of mutations at the cleavage site. The genetically modified embryos are transferred to a surrogate mother, which gives birth to founder animals that carry the desired knockout mutation.
challenges. Targeted gene inactivation, as applied in the monkeys, could be used to treat disorders caused by heterozygous gain-of-function or dominant-negative mutations. (A dominant-negative mutation results in the interference of the mutant protein with the function of the nonmutant protein.) Diseases of the hematopoietic system, such as primary immunodeficiencies caused by gain-of-function mutations in STAT1, encoding the signal transducer and activator of transcription 1 protein, is an example of such a disorder. Although the study of CRISPR–Cas9 and TALEN is still in its early stages, many major breakthroughs in improving these two nuclease platforms have recently been made, including modifications to improve the specificity of targeting to a specific site in the genome. Off-target cleavage — potentially resulting in mutagenic events that lead to a malignant phenotype — is a major concern, especially if it occurs in multipotent stem cells destined to be transplanted in patients. Hence, the specificity of engineered nucleases will be the key factor in the translation of this new line of gene therapy into clinical practice. The first human application of designer endonucleases was recently described in the Journal.5 Zinc-finger nucleases were used to disrupt the gene encoding the human immunodeficiency virus (HIV) coreceptor CCR5 in autologous CD4 T cells isolated from, and then transfused back into, persons with HIV infec-
tion, with the goal of engineering cellular resistance to HIV. The study showed that infusion of CCR5-modified T cells was safe, although the sample was small (12 patients) and follow-up was limited (36 weeks); further tests of safety involving a larger sample and long-term followup have yet to be reported. To maintain a durable therapeutic effect, it may be necessary for gene editing to be targeted to the DNA of stem cells. Postnatal somatic stem cells, such as hematopoietic stem cells, are the most accessible cell type for targeting, but increasingly successful protocols for differentiating induced pluripotent stem cells give hope that an approach involving the harvesting of somatic cells, induction of pluripotency, and correction of mutation, followed by differentiation and transplantation, will become a reality.6 The successful genetic editing of one-cell primate embryos raises ethical issues that go beyond the balance between the scientific value of transgenic monkey models in understanding human disease and concerns about their creation. These studies bring us one step closer to the potential for manipulating genes in human embryos. With an open attitude and the will to promote discussions of ethics, scientists developing DNA-editing technologies have the opportunity to engage in and enrich public discourse, in addition to working toward the alleviation of human suffering from genetic diseases. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. From the Center for Chronic Immunodeficiency (T.C., S.E.), the Institute for Cell and Gene Therapy (T.C.), and the Department of Pediatrics and Adolescent Medicine (S.E.), University Medical Center Freiburg, Freiburg, Germany. 1. Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cy-
nomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014;156:836-43. 2. Liu H, Chen Y, Niu Y, et al. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell 2014; 14:323-8. 3. de Souza N. Primer: genome editing with engineered nucleases. Nat Methods 2012;9:27. 4. Genetic microsurgery for the masses. Science 2013;342: 1434-5. 5. Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014;370:901-10. 6. Garate Z, Davis BR, Quintana-Bustamante O, Segovia JC. New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells. Hum Gene Ther 2013;24:571-83. DOI: 10.1056/NEJMcibr1403629 Copyright © 2014 Massachusetts Medical Society.
n engl j med 370;24 nejm.org june 12, 2014
The New England Journal of Medicine Downloaded from nejm.org at UNIVERSITY OF PITTSBURGH on June 12, 2014. For personal use only. No other uses without permission. Copyright © 2014 Massachusetts Medical Society. All rights reserved.
2345
