The Time Is Ripe for Somatic Genome Editing: NIH Program to Strengthen Translation
Authors: Boris Fehse, Ulrike Abramowski-Mock
Journal: Molecular Therapy (2018)

Targeted genome editing ("genomic surgery") has been the holy grail of gene therapists for more than five decades. In the 1980s, the first proof-of-concept was obtained that cellular DNA repair mechanisms such as homologous recombination—evolved to protect genome integrity—can be hijacked to deliberately introduce changes into the genome. Homologous recombination is a copy-paste mechanism that, in the natural situation, uses the ideally unaffected second copy (allele) of a given genomic locus as a repair matrix to replace the mutated area. Mario R. Capecchi, Oliver Smithies, and Martin J. Evans were the first to exploit this mechanism to alter the genomes of mammalian including human cells.1Smithies O. Gregg R.G. Boggs S.S. Koralewski M.A. Kucherlapati R.S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination.Nature. 1985; 317: 230-234Crossref PubMed Scopus (670) Google Scholar, 2Capecchi M.R. Altering the genome by homologous recombination.Science. 1989; 244: 1288-1292Crossref PubMed Scopus (1611) Google Scholar Their research was awarded the Nobel Prize in 2007. An alternative DNA repair mechanism, non-homologous end-joining (NHEJ)3Moore J.K. Haber J.E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.Mol. Cell. Biol. 1996; 16: 2164-2173Crossref PubMed Scopus (596) Google Scholar is more efficient and less demanding (e.g., no template required), which often makes it the preferred choice of cells. However, NHEJ initially seemed less attractive for genome editing because it is error-prone and therefore results in a huge variety of different mutations in repaired cells. To make use of the above repair mechanisms for genome editing, (double-) strand breaks had to be introduced at the locus of interest with high precision. Most "classical" DNA-cutting enzymes usually referred to as restriction enzymes (based on their function in the bacterial innate immune system) were not suitable due to their short recognition sequences. However, single bacterial nucleases called meganucleases recognized particularly long DNA stretches, such as I-SceI with a DNA-binding site of 18 nucleotides.4Plessis A. Perrin A. Haber J.E. Dujon B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus.Genetics. 1992; 130: 451-460PubMed Google Scholar Since there is a high probability that an 18-nucleotide (nt) stretch is only once present in the human genome, such meganuclease would, in principle, be suitable for targeted cutting.5Rouet P. Smih F. Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.Mol. Cell. Biol. 1994; 14: 8096-8106Crossref PubMed Scopus (615) Google Scholar Unfortunately, there was only a very tiny chance that any site of interest for genome editing contained the recognition site of one of the few available meganucleases. This stimulated the idea to change the recognition sequence of meganucleases by sophisticated protein engineering.6Smith J. Grizot S. Arnould S. Duclert A. Epinat J.C. Chames P. Prieto J. Redondo P. Blanco F.J. Bravo J. et al.A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.Nucleic Acids Res. 2006; 34: e149Crossref PubMed Scopus (246) Google Scholar Whereas the approach turned out to be a very difficult endeavor (since the enzymatic and the recognition functions of these enzymes are combined within the same protein domains), it already illustrated the concept of creating high-specificity "designer nucleases." To make this concept more broadly applicable, the two main functions of a designer nuclease (sequence-specific), DNA binding and nuclease activity, had to be separated into two distinct protein domains. Thankfully, nature has provided us with functional examples of such enzymes, namely type-IIS endonucleases. Thus the task was obvious, although not easy: the DNA binding domain of a type-IIS endonuclease (such as Fok I) had to be replaced with an artificial domain recognizing the sequence of interest. This became possible when the structure of the so-called Zinc fingers, DNA-binding finger-like protein domains containing a Zinc ion, was revealed.7Thiesen H.J. Bach C. Determination of DNA binding specificities of mutated zinc finger domains.FEBS Lett. 1991; 283: 23-26Crossref PubMed Scopus (13) Google Scholar This laid the basis for ground-breaking work on the generation of chimeric, DNA-binding proteins with different functions, including the first designer nucleases.8Kim Y.G. Cha J. Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc. Natl. Acad. Sci. USA. 1996; 93: 1156-1160Crossref PubMed Scopus (1403) Google Scholar Zinc-finger nucleases (ZFNs) were not only the first designer nucleases to be used for various research applications, they also opened the way for genome editing into the clinics.9Tebas P. Stein D. Tang W.W. Frank I. Wang S.Q. Lee G. Spratt S.K. Surosky R.T. Giedlin M.A. Nichol G. et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N. Engl. J. Med. 2014; 370: 901-910Crossref PubMed Scopus (1008) Google Scholar However, the generation of ZFNs has remained difficult, cumbersome, and expensive, preventing broad distribution of this technology. Determination of the structure of another class of DNA-binding proteins, transcription-factor like effectors (TALEs)10Boch J. Scholze H. Schornack S. Landgraf A. Hahn S. Kay S. Lahaye T. Nickstadt A. Bonas U. Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1906) Google Scholar, 11Moscou M.J. Bogdanove A.J. A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1457) Google Scholar renewed the enthusiasm in the genome-editing field and led Nature Methods to name the technique "Method of the Year" in 2011. TALEs were isolated from plant bacteria, which use them to reprogram the transcription machinery of infected plant cells. They had several advantages, including a 1-to-1 code for DNA recognition (one monomer of the DNA-binding domain recognizes one nucleotide) and long target sequences resulting in very high specificity. However, their main benefit was the ease of cloning (based on various elegant strategies, discussed in Schmid-Burgk et al.12Schmid-Burgk J.L. Schmidt T. Hornung V. Ligation-independent cloning (LIC) assembly of TALEN genes.Methods Mol. Biol. 2015; 1239: 161-169Crossref PubMed Scopus (9) Google Scholar) shortening the time from the idea to the protein from several months (for ZFNs) to a few weeks for TALE nucleases (TALENs). Thus, since the early 2010s, labs all over the world have been able to develop high-efficiency TALENs for multiple target genes, including therapeutically relevant ones, such as CCR5.13Mock U. Machowicz R. Hauber I. Horn S. Abramowski P. Berdien B. Hauber J. Fehse B. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.Nucleic Acids Res. 2015; 43: 5560-5571Crossref PubMed Scopus (96) Google Scholar As a consequence, translation toward the clinics was much faster for TALENs, with the first clinical application appearing in 2015.14Qasim W. Zhan H. Samarasinghe S. Adams S. Amrolia P. Stafford S. Butler K. Rivat C. Wright G. Somana K. et al.Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells.Sci. Transl. Med. 2017; 9: eaaj2013Crossref PubMed Scopus (575) Google Scholar Very soon after, TALENs were pushed aside by the new shooting star, the CRISPR/Cas system.15Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9321) Google Scholar Despite its complicated name (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated [protein]), this new tool is extremely easy to be applied, which has made all alternative means of genome editing look very old-fashioned. CRISPR/Cas and subsequently developed similar systems have been derived from bacterial adaptive immune systems.16Gasiunas G. Barrangou R. Horvath P. Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc. Natl. Acad. Sci. USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1657) Google Scholar To efficiently recognize and destroy genetic enemies, such as bacteriophages, bacteria use the CRISPR/Cas system to memorize parts of the genetic information of the offender by including short DNA pieces into their own genome (in the CRISPR array) and expressing them as short RNA molecules (CRISPR or crRNAs). Similarly to our antibodies, these short crRNAs will specifically recognize the phages should they infect the bacteria a second time. Binding of the phage DNA triggers a specific nuclease (Cas9) to hack the viral genome into pieces. The interesting story of the development of CRISPR/Cas and the pioneering heroes has recently been told by Eric Lander.17Lander E.S. The Heroes of CRISPR.Cell. 2016; 164: 18-28Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar The huge advantage of CRISPR/Cas9 is based on the mechanism of DNA binding, which, in contrast to all previous designer nucleases, is mediated by an RNA molecule. In 2012, Jinek et al.15Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9321) Google Scholar showed that just two RNA molecules (crRNA and tracrRNA) and only one of the Cas proteins (Cas9) are sufficient for targeted genome disruption in mammalian cells. Moreover, the two RNAs could be fused into one chimeric molecule,15Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9321) Google Scholar now referred to as single-guide RNA (sgRNA). Soon thereafter it was shown that CRISPR/Cas9 facilitated genome editing in human cells.18Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9977) Google Scholar, 19Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6423) Google Scholar, 20Jinek M. East A. Cheng A. Lin S. Ma E. Doudna J. RNA-programmed genome editing in human cells.eLife. 2013; 2: e00471Crossref PubMed Scopus (1495) Google Scholar DNA binding by a sequence-specific RNA rather than a protein domain has a huge advantage—no novel protein has to be designed for any new application. Instead, a short DNA serving as a template for the RNA transcription is sufficient. Since synthesis of the necessary DNA oligonucleotides can be done overnight, a new "designer nuclease" with the desired specificity can be obtained in a couple of days rather than weeks. Moreover, and also in contrast to previous designer nucleases, CRISPR/Cas allows multiplexing (i.e., simultaneous cutting of the target genome at different places),18Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9977) Google Scholar, 19Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6423) Google Scholar, 21Wang H. Yang H. Shivalila C.S. Dawlaty M.M. Cheng A.W. Zhang F. Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153: 910-918Abstract Full Text Full Text PDF PubMed Scopus (2584) Google Scholar using one and the same enzyme directed by several guide RNAs. The ease and speed of designing and getting any new sgRNA/Cas9 complex against a given target implies that even malfunction or high off-target activity of a single gRNA does not represent a problem anymore, since in most cases it can readily be replaced by an alternative one. Thus, the whole system, all its derivatives and relatives can permanently be optimized, which is an enormous benefit not the least when considering clinical applications. The best indicator for the success of CRISPR/Cas has been the extremely fast distribution into obviously all areas of wet-lab life-technology research. Moreover, translation into clinical application in somatic gene therapy was even faster than with TALENs. The first patients were treated with "crispered" immune cell in China in 2016 (commented in Nature22Cyranoski D. CRISPR gene-editing tested in a person for the first time.Nature. 2016; 539: 479Crossref PubMed Scopus (266) Google Scholar). Meanwhile, at least 10 clinical CRISPR studies are ongoing in China, so far all with edited blood cells; several others are probably just around the corner in many countries. Moreover, there are an equal number of studies ongoing in different entities with ZFNs and TALENs, which of course had a head start in development of several years. A few studies have focused on monogenic diseases, the "natural" target of genome editing. However, most approaches so far have aimed at improving immunotherapy in cancer or protecting cells from HIV (Table 1).Table 1Gene Editing Clinical Trials (Source: ClinicalTrials.gov, January 31, 2018)Target GeneKnockin / AdditionConditionDeliveryCountryNCT NumberZFNCCR5HIVex vivo AV, mRNA- or not statedUSANCT00842634NCT01044654NCT01543152NCT02225665NCT02388594NCT02500849HPV16 or 18 E7HPV-related malignant neoplasmin vivo suppositoryaContaining 500μg ZFN or TALEN (likely to be DNA).ChinaNCT02800369albuminFIX, IDS or IDUAhemophilia B; MPS II or Iin vivo AAV 2/6USANCT02695160NCT03041324NCT02702115TALENTCRα / CD5214Qasim W. Zhan H. Samarasinghe S. Adams S. Amrolia P. Stafford S. Butler K. Rivat C. Wright G. Somana K. et al.Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells.Sci. Transl. Med. 2017; 9: eaaj2013Crossref PubMed Scopus (575) Google ScholarαCD19-CARrelapsed/refractory B-ALLex vivo TALEN- mRNA-CAR-LVV23Cai T. Galetto R. Gouble A. Smith J. Cavazos A. Han L. Zhang Q. Kuruvilla V.M. Konoplev S.M. Neelapu S.S. et al.Pre-clinical studies of allogeneic anti-CD123 CAR T-cells for the therapy of blastic plasmacytoid dendritic cell neoplasm (BPDCN).Blood. 2017; 130: 2625Google ScholarEurope and USANCT02808442NCT02746952TCRαbhttp://www.cellectis.comαCD123-CARAML or BPDCNUSANCT03190278NCT03203369HPV16 E6 / E7HPV-related malignant neoplasmin vivo suppositoryaContaining 500μg ZFN or TALEN (likely to be DNA).ChinaNCT03226470TALEN and CRISPR/CasHPV16 E6/E7HPV18 E6/E7HPV-related malignant neoplasmin vivo plasmid in gelChinaNCT03057912CRISPR/CasCCR5HIVex vivo delivery not statedChinaNCT03164135TCR / B2MαCD19-CARB cell leukemia / lymphomaex vivoCRISPR- RNA-cDelivery of Cas9 not explicitly described.CAR-LVVChinaNCT03166878PD-1solid tumorsdMetastatic non-small cell lung cancer, invasive bladder cancer stage IV, hormone refractory prostate cancer, metastatic renal cell carcinoma, esophageal cancer, and EBV-associated malignancies.ex vivo delivery not statedChinaNCT02793856NCT02863913NCT02867345NCT02867332NCT03081715NCT03044743not statedeTarget genes not stated (likely to be TCR and B2M as in study NCT03166878).αCD19/20 or αCD19/22-CARsB cell leukemia / lymphomaex vivodelivery not statedChinaNCT03398967TCRα+β / PD-1NY-ESO-1-TCRMM, melanoma synovial sarcoma liposarcomafMyxoid or round cell liposarcoma.ex vivo CRISPR- RNA-cDelivery of Cas9 not explicitly described.TCR-LVVUSANCT03399448n.a.gLikely to be CRISPR/Cas.PD-1αCD19-CARALL Burkitt lymphomaex vivo delivery not statedChinaNCT03298828ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AV, adenoviral vector; BPDCN, blastic plasmacytoid dendritic cell neoplasm; CAR, chimeric antigen receptor; FIX, factor IX; HIV, human immunodeficiency virus; HPV, human papilloma virus; = electroporation; IDS, iduronate 2-sulfatase; IDUA, α-L-iduronidase; LVV, lentiviral vector; MM, multiple myeloma; MPS, mucopolysaccharidosis; n.a., not available.a Containing 500μg ZFN or TALEN (likely to be DNA).b http://www.cellectis.comc Delivery of Cas9 not explicitly described.d Metastatic non-small cell lung cancer, invasive bladder cancer stage IV, hormone refractory prostate cancer, metastatic renal cell carcinoma, esophageal cancer, and EBV-associated malignancies.e Target genes not stated (likely to be TCR and B2M as in study NCT03166878).f Myxoid or round cell liposarcoma.g Likely to be CRISPR/Cas. Open table in a new tab ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AV, adenoviral vector; BPDCN, blastic plasmacytoid dendritic cell neoplasm; CAR, chimeric antigen receptor; FIX, factor IX; HIV, human immunodeficiency virus; HPV, human papilloma virus; = electroporation; IDS, iduronate 2-sulfatase; IDUA, α-L-iduronidase; LVV, lentiviral vector; MM, multiple myeloma; MPS, mucopolysaccharidosis; n.a., not available. At the same time, none of the available genome-editing tools is perfect. Their use in devastating diseases is obviously well-founded, but application in less severe conditions will require thorough assessment of the risk/benefit ratio. To address these questions, the NIH Common Fund program has launched a new initiative called Somatic Cell Genome Editing (https://commonfund.nih.gov/editing). The main aim of this program is the development of quality tools to perform effective and safe genome editing in human patients. As the NIH states, "These research tools will be made widely available to the research community to reduce the time and cost required to develop new therapies." The four tasks defined by the program reflect the main challenges of broad introduction of somatic genome editing into clinical practice: (1) the availability of a suitable tool for a given target site; (2) the delivery problem; (3) the need for assays to assess both safety and efficacy; and (4) the broad access to all these tools. Altogether, this is very timely initiative, which surely will be acclaimed by all researchers in the field. Therefore, Molecular Therapy, the official flagship journal of the ASGCT, is very happy to support and promote this project. The authors are inventors on a patent for a specific TALEN (CCR5-Uco-TALEN) applied for by the UMC Hamburg-Eppendorf. B.F. is consulting AGCT, a biotech active in the application of genome editing. The authors' research on the clinical implications of genome editing has been supported by the German Federal Ministry of Education and Research (BMBF) within the collaborative project GenE-TyPE (#01GP1610B) and by the German Center for Infection Research (DZIF).