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BIOPHARMANALYSES
TrendChart
oN GENE Editing
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contents
March 2018, 2nd

DISRUPTIVE TECHNOLOGIES
● CRISPR RNAs trigger innate immune responses in human cells
● i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases
● Long Terminal Repeat CRISPR-CAR coupled ‘universal’ T cells mediate potent anti-leukemic effects

GENOME EDITING AND DISEASES
● Cholesterol halved by CRISPR/Cas9 base editing 
● One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system.
● Precise excision of the CAG tract from the Huntingtin gene by Cas9 nickases 
● Efficient in vivo liver-directed gene editing using CRISPR/CAS9 

CLINICAL TRIALS
●   Sangamo announces U.K. authorization of clinical trial evaluating Zinc Finger Nuclease in vivo genome editing treatment for Hemophilia B

INDUSTRIAL LANDSCAPE - AGREEMENTS
●  A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing
● Inscripta raises 55.5MUS$ to advance gene-editing technology
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FEATURE STORY
CRISPR RNAs trigger innate immune responses in human cells
In a recent publication, Korean investigators report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5'-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.
The results appeared in February 22nd online issue of Genome Res  ( https://genome.cshlp.org/content/28/3/367 )
Related Informations/Publications
  Ther Adv Med Oncol. 2018 Feb 5;10:1758834018755089. CRISPR-Cas9: a promising genetic engineering approach in cancer research. Ratan ZA et al. Sungkyunkwan University, 2066 Seobu-ro, Suwon 16419, Korea. Results /Comments : This article reviews the concept of CRISPR-Cas, its application and related advantages in oncology.
Link : Abstract , Full Text  .
•   Eur J Immunol . 2018 Feb 7. doi: 10.1002/eji.201747131. CRISPR/Cas9: A tool for immunological research. Hochheiser K et al. University of Melbourne, Parkville, Victoria, Australia
Results / Comments : The first clinical trials that use CRISPR/Cas9 and discuss potential future applications are discussed. .
Link : Abstract
i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases
Japanese researchers have recently presented a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.
The results appeared in February 26th online issue of Genome Biol .
Related informations/publications
Nat Commun . 2018 Jan 29;9(1):412. Streamlined ex vivo and i n vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Yoon Y et al. University of Massachusetts Medical School, Worcester, MA, 01655, USA.
Results / Comments : The approach presented simplifies the generation of genetically modified mice and, more importantly, opens the door for streamlined gene editing in other mammalian species.
Link : Abstract Text
Eur J Hum Genet . 2018 Jan;26(1):1-11. One small edit for humans, one giant edit for humankind? Points and questions to consider for a responsible way forward for gene editing in humans. Howard HC et al. Uppsala University, Uppsala, Sweden
Link : Abstract .
Long Terminal Repeat CRISPR-CAR coupled ‘universal’ T cells mediate potent anti-leukemic effects
Gene editing can be used to overcome allo-recognition which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such ‘universal’ T cells expressing chimeric antigen receptors (CAR) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). While relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. Investigators have described a self-inactivating lentiviral ‘terminal’ vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3’ long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogenous (>96%) CAR+ (>99%) TCR- populations by automated magnetic separation. Molecular analyses, including NGS, WGS and Digenome-seq verified on-target specificity with no evidence of predicted off-target events.
The results appeared in February 28th online issue of Mol Ther  
Related informations/publications
JJ Exp Med . 2018 Feb 7. pii: jem.20171626. doi: 10.1084/jem.20171626. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. Seki A, Rutz S. Department of Cancer Immunology, Genentech ( South San Francisco, CA, USA.
Results /Comments : The publication describes an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation.
Link : Abstract .
Protein Cell . 2017 Sep;8(9):634-643. Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9. Ren J, Zhao Y. University of Pennsylvania, Philadelphia, PA, USA.
Link : Abstract . Full Text
• DEC 2017 : Mustang Bio  Launches CRISPR/Cas9 CAR-T Collaborations with Harvard, BIDMC
Link : Press Release .
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GENOME EDITING AND DISEASES
Cholesterol halved by CRISPR/Cas9 base editing
Naturally occurring loss-of-function mutations in ANGPTL3 (angiopoietin-like 3) are associated with reduced blood triglycerides, low-density lipoprotein cholesterol, and risk of coronary heart disease, with no apparent adverse health consequences, making ANGPTL3 a compelling therapeutic target. American investigatores assessed whether base editing, a variation on CRISPR-Cas9 genome editing that does not require DNA double-strand breaks, could be used in vivo to introduce loss-of-function mutations into ANGPTL3 and reduce blood lipid levels. Base editor 3 (BE3) can introduce cytosine-to-thymine changes at desired sites in the genome, eg, nonsense mutations, into Pcsk9 (proprotein convertase subtilisin/kexin type 9) in mice. The results appeared in February 26th online issue of Circulation .
Related Informations / Publications 
  Curr Atheroscler Rep . 2017 Jul;19(7):32. Treatment of Dyslipidemia Using CRISPR/Cas9 Genome Editing. Chadwick AC, Musunuru K. University of Pennsylvania, Philadelphia, PA, USA.
Link : Abstract
•  Hepatol Commun . 2017 Nov;1(9):886-898. CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells. Omer L et al. University of Louisville School of Medicine, Louisville, KY, USA.
Results / Comments : The genetic correction restored LDLR-mediated endocytosis in FH-HLCs and demonstrates the proof-of-principle that CRISPR-mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level.
Link : Abstract . Full Text .
One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system
Thalassemia  is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. Tahi researchers have reported genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. They used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression.
The results appeared in February 28th online issue of Stem Cell Res Ther
Related Informations / Publications
• Nat Protoc . 2018 Feb;13(2):358-376. CRISPR/Cas9 genome editing in human hematopoietic stem cells. Bak RO et al. Stanford University, Stanford, California, USA.
 Link : Abstract . Full Text
 Blood Cells Mol Dis . 2018 Jan 3. pii: S1079-9796(17)30429-1. Gene therapy and gene editing strategies for hemoglobinopathies. Lidonnici MR, Ferrari G. San Raffaele Scientific Institute, Milan, Italy.
Link : Abstract
Precise excision of the CAG tract from the Huntingtin gene by Cas9 nickases
Huntington's disease is caused by the abnormal repetition of a specific DNA sequence at the tail end of the huntingtin gene. This defective mutant gene causes production of a toxic protein that progressively accumulates and damages the patient's neurons. The disease usually begins in a patient's 30s or 40s and, in the decades after disease onset, patients gradually lose the ability to move, talk and even think. There is currently no cure for Huntington's disease. But researchers have tried many methods to silence the defective gene. This includes interrupting production of the toxic protein through DNA- and RNA-based approaches. Most recently, researchers have also begun work with one of the most promising gene-editing tools to date -- the CRISPR/Cas9 system, which is far easier, faster and more specific than past tools. But it is still the early days of medical applications of CRISPR/Cas 9, which was only discovered in 2012. To make sure that this technique is as safe and effective as possible, a Polish group group has been testing out a new variant of the Cas9 protein component in cellular models from a Huntington's patient. This version of Cas9 was recently designed to act as a nickase -- an enzyme that cuts just one DNA strand instead of two, which increases the precision with which Cas9 can edit specific sequences of DNA. The results appeared in February 26th online issue of Front Neurosci
Related Informations / Publications
  J Clin Invest . 2017 Jun 30;127(7):2719-2724. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington's disease. Yang S et al. Emory University School of Medicine, Atlanta, Georgia, USA. Results / Comments : Non-allele-specific CRISPR/Cas9-mediated gene editing could be used to efficiently and permanently eliminate polyglutamine expansion.
Link : Abstract . Full Text
•  J Huntingtons Dis. 2017;6(1):19-31. doi: 10.3233/JHD-160222. Modern Genome Editing Technologies in Huntington's Disease Research. Malankhanova TB et al. The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Link : Abstract . Full Text .  
Efficient in vivo liver-directed gene editing using CRISPR/CAS9
In vivo tissue-specific genome editing at the desired loci is still a challenge. Belgian investigators now report that AAV9-delivery of truncated guide RNAs (gRNAs) and Cas9 under the control of a computationally designed hepatocyte-specific promoter lead to liver-specific and sequence-specific targeting in the mouse factor IX (FIX) gene. The efficiency of in vivo targeting was assessed by T7E1 assays, site-specific Sanger sequencing and deep sequencing of on-target and putative off-target sites. Though AAV9 transduction was apparent in multiple tissues and organs, Cas9 expression was restricted mainly to the liver, with only minimal or no expression in other non-hepatic tissues. Consequently, the indel frequency was robust in the liver (up to 50%) in the desired target loci of the FIX gene, with no evidence of targeting in other organs or other putative off-target sites. This resulted in a substantial loss of FIX activity and the emergence of a bleeding phenotype, consistent with hemophilia B . The in vivo efficacy of the truncated gRNA was as high as that of full-length gRNA. The results appeared in February 27th online issue of Mol Ther .  
Related Informations / Publications
•  Blood Adv. 2017 Oct 16;1(23):2019-2031. Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors. Meliani A, et al. University Pierre and Marie Curie-Paris 6 and INSERM U974, Paris, France
Link : Abstract . Full Text .  
• DEC 2017 : Spark Therapeutics  and Pfizer Announce Longer-term preliminary Data Showing Consistent and Sustained Factor IX Levels in Hemophilia B at 59th American Society of Hematology (ASH) Annual Meeting and Exposition
Link : Press Release
• DEC 2017 : BioMarin Doses First Patient in Global GENEr8-1 Phase 3 Study of Valoctocogene Roxaparvovec Gene Therapy for Severe Hemophilia A
Link: Press Release  
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CLINICAL TRIALS
Sangamo announces U.K. authorization of clinical trial evaluating Zinc Finger Nuclease in vivo genome editing treatment for Hemophilia B
Sangamo Therapeutics of Richmond, Calif., said on February 28th that the Medicines and Healthcare Products Regulatory Agency of the U.K. granted a clinical trial authorization (CTA) allowing Sangamo to enroll patients in its ongoing phase I/II trial testing SB-FIX, a zinc finger nuclease-mediated in vivo genome editing treatment for hemophilia B. Sangamo plans to add clinical sites in the U.K. to the trial later this year. Preliminary safety and efficacy data will be determined in adults before adolescents ages 12 to 17 will be enrolled in the trial. Sangamo also noted plans to file CTAs for SB-318 and SB-913, in vivo genome editing treatments for mucopolysaccharidosis type I and type III, respectively. For further info  
Related Informations / Publications
• OCT 2017: Sangamo And Bioverativ Announce FDA Acceptance Of IND Application For ST-400 -- A Gene-Edited Cell Therapy Candidate -- To Treat Beta-Thalassemia.
Link: Press Release  
• AUG 2017: Sangamo And Pfizer Announce First Patient Receives Treatment In Phase 1/2 Clinical Trial Evaluating SB-525 Investigational Gene Therapy For Hemophilia A.
 Link: Press Release  
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INDUSTRIAL LANDSCAPE AND AGREEMENTS
A Single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing
Intellia Therapeutics of Cambridge, Mass., said on February 27th that recently published preclinical data show that the lipid nanoparticle (LNP) delivery of Cas9 mRNA and sgRNA resulted in 97 percent reduction in mouse transthyretin protein levels in the liver, and the reduction was sustained for at least 12 months. The publication also documents that CRISPR/Cas9 components were undetectable in mice within three days after administration of Intellia’s LNP delivery system. Researchers further demonstrated that Intellia’s LNP technology is a similarly robust and effective delivery method for CRISPR/Cas9-mediated knockdown in rats, a higher rodent species.
The results appeared in February 27th online issue of Cell Rep .
Related Informations / Publications
•  DEC 2017 : Intellia Therapeutics Announces New, Robust Genome Editing Data for Sickle Cell Disease at the American Society of Hematology Meeting
Link: Press Release  
•  SEP 2017 : Intellia Therapeutics Demonstrates Sustained and Durable Genome Editing with CRISPR/Cas9 in One-Year Animal Study
Results / Comments: One-year data demonstrate sustained TTR protein reduction of approximately 97 percent, corresponding to 70 percent liver editing, following a single in vivo systemic dose in mice.
Link: Press Release
Inscripta raises 55.5MUS$ to advance gene-editing technology
Inscripta , a leading gene-editing technology company, announced on February 28th that it has closed a $55.5 million Series C funding round led by Mérieux Développement and Paladin Capital Group. Additional participants include all existing Inscripta investors: Venrock, Foresite, MLS Capital, and NanoDimension. The new funding comes on the heels of the company’s release of its first CRISPR enzyme (MAD7), and will accelerate Inscripta’s development and commercialization of gene-editing tools, including instruments, reagents, and software, and grow the company’s expert team.
For further info 
Related Informations / Publications
•  DEC 2017 : Inscripta Releases New, Unique CRISPR Gene-Editing Enzyme; Free to Use for All Researchers Link: Press Release  
•  FEB 2017: Muse bio (now Inscripta) secures $23M Series B financing led by Venrock
Link: Press Release
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