Unedited Cells Research Articles

Abstract 10268: Establishment of a Hypoimmunogenic Human Induced Pluripotent Stem Cell Line Which Allows in Vivo Tracing of Ventricular Cardiomyocytes

Introduction: In recent years, human induced pluripotent stem cells (hiPSCs) have emerged as a promising source for cardiac cell therapy. However, immune rejection and insufficient methods for cell fate tracing compromise the assessment of graft efficiency and survival. Hence, we sought to establish a hiPSC line for regenerative therapies which combines hypoimmunogenic and in vivo traceable features. Methods: A hiPSC line was modified using CRISPR/Cas9 technology to first insert a tdTomato reporter into the MLC2v gene and in a second step to replace exons 2 and 3 of the B2M gene with a neomycin resistance cassette, designated B2M-KO. MHC-I surface expression was evaluated by flow cytometry analysis. The immune response of cytotoxic CD8+ T cells and natural killer (NK) cells against the B2M-KO and wild-type hiPSCs was evaluated with a lactate dehydrogenase-based cytotoxicity assay. Immunocytochemistry and qRT-PCR expression analyses were performed to study the cardiomyogenic capacity of B2M-KO hiPSCs during directed cardiac differentiation. Results: The homozygous disruption of the B2M gene was confirmed by qRT-PCR and flow cytometry. However, mRNA expression of essential cardiac transcription factors ( NKX2.5, TBX5 ) and important structural genes ( MLC2a, MLC2v ) during cardiac differentiation was not affected. By contrast, CD8+ T cell-mediated cytotoxicity against B2M -deficient hiPSCs was dramatically reduced compared to wild-type hiPSCs (p<0.002) while NK cell-dependent lysis remained unaffected. The tdTomato fluorescence in differentiating B2M -deficient hiPSCs was generally detectable around day 20 of the directed cardiac differentiation protocol and increased over time. These cells concomitantly also showed expression of MLC2v . Finally, immunocytochemistry confirmed expression of MLC2a, MLC2v and α-ACTININ similar to unedited cells after 30 days of cardiac differentiation. Conclusions: Genetic manipulation of MHC-I surface expression is an efficient strategy to reduce T cell-mediated immune rejection, yet unable to decrease NK cell-mediated cytotoxicity. The generated B2M-KO hiPSC line exhibits hypoimmunogenic properties and allows post-transplantational in vivo tracking of ventricular cardiomyocytes.

Knock out of CD123 or CLL-1 By CRISPR-Cas9 Editing from Human Hematopoietic Stem Cell Transplants Provide New Possibilities for Increasing Therapeutic Index and Safety for AML Treatment

▪Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis and the most common form of acute leukemia in adults. Most patients with AML relapse despite intensive chemotherapy. Allogeneic hematopoietic cell transplant (HCT) has become the standard of care for patients with intermediate or adverse genetics, with >3,500 transplants performed annually in the US. However, leukemia relapse post-HCT occurs in ~40% of these patients with a 2-year survival rate at <20%, necessitating new approaches to reduce relapse and improve overall outcomes.Antigen-specific immunotherapies require cell surface antigens to be uniquely expressed on cancer cells to minimize “on-target, off-tumor” toxicity. CD123 and CLL-1 are highly expressed on normal myeloid cells, thus impeding the use of therapies targeting these antigens due to myelotoxicity. To circumvent such toxicity, we aim to create CD123 or CLL-1 negative human hematopoietic stem and progenitor cells (hHSPCs) for HCT such that this can be combined with subsequent use of targeted therapy against these antigens to prevent post-HCT relapse. Here, we present the pre-clinical evaluation of engineered hHSPCs, derived from mobilized peripheral blood of healthy donors, where CD123 or CLL-1 proteins were ablated by CRISPR/Cas9 gene editing. We have identified highly efficient guide RNAs that result in >80% on-target editing and can achieve greater than 90% biallelic gene knockout (KO). Deep sequencing followed by hybrid capture of up to 1000 potential off-target sites predicted to contain a maximum of 5 mismatches revealed minimal to no detectable off-target editing events. CD123 or CLL-1 KO hHSPCs showed >85% cell viability post-editing. Loss of CD123 or CLL-1 protein did not impact the differentiation of hHSPCs into granulocytic, monocytic, or erythroid lineages in vitro. Additionally, myeloid cells derived from CD123 or CLL-1 KO hHSPCs also retained their function, demonstrating similar phagocytotic capacity and the production of inflammatory cytokines in response to TLR agonists compared to unedited control cells. Importantly, CD123 or CLL-1 KO hHSPCs xenotransplanted into NSG or NBSGW mice showed no defect in long-term engraftment (human chimerism in NBSGW mice 16 weeks post transplant: 92.3±5.1% for CD123 KO, 91.2±4.1% for CLL-1 KO, and 93.4±3.1% unedited hHSPC engrafted mice, n=15 each). This in vivo model confirmed no observable impact on multilineage differentiation after CD123 or CLL-1 deletion either, supported by equal distribution of 10 hematopoietic lineages between groups. Bone marrow analyses revealed persistence of high gene editing frequency after 16 weeks (71±10% from CD123 KO and 86±6% from CLL-1 KO engrafted mice, n=15 each, compared to original 83% input CD123 KO and 86% input CLL-1 hHSPCs). Sorted subpopulations, including granulocytes, monocytes, dendritic cells, and mast cells, from the bone marrow similarly retained high levels of editing. Together these data suggest that there was no counter-selection against CD123 or CLL-1 KO cells, no outgrowth of any cells with particular edits or hematopoietic lineages to warrant tumorigenic concerns. Most notably, proof-of-concept experiments showed that cells depleted of CD123 or CLL-1 protein are indeed protected from the cytotoxicity of CD123 or CLL-1 targeted CAR-T cells.In conclusion, we demonstrate that CD123 or CLL-1 negative human HSPCs can successfully carry out functional hematopoiesis that is resistant to CD123 or CLL-1 targeted therapies. Our findings provide a next-generation HCT strategy that supports the safe and effective use of antigen-directed immunotherapy treatments for patients with AML. DisclosuresLuo: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Angelini: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Krishnamurthy: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lisle: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Isik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ghdossi: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Pettiglio: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hazelbaker: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Tavares: Vor Biopharma: Ended employment in the past 24 months. Nikam: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Paik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lydeard: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

Base Editing Repairs the HbE Mutation Restoring the Production of Normal Globin Chains in Severe HbE/β-Thalassemia Patient Hematopoietic Stem Cells and Erythroid Cells

HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal.We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D).To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing.Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiplex Engineering of Human CD34+ HSPCs Enables Dual Gene Knock-out While Maintaining High Engraftment Potential and Safety

Genome engineering of allogeneic hematopoietic stem cell transplants (HSCT), in which leukemia-specific cell surface antigens are removed by editing from the donor graft, may provide high therapeutic index and safety in combination with post-transplant targeted therapeutics. This strategy allows the therapeutics to specifically target leukemic cells by sparing the gene-edited graft, and thereby enable the next generation of HSCTs for treatment of acute myeloid leukemia (AML). So far, no single target antigen is known to be expressed in 100% of AML blasts, leukemic stem cells, or even in affected patients. Therefore, use of combinatorial therapies targeting multiple antigens may represent a potential paradigm shift in the standard of care for AML. Multiplex editing of HSCT in which two or more target antigens are removed from the same cell would enable combinatorial targeting while simultaneously protecting the engineered graft from on-target, off-tumor toxicity. Here we report proof-of-concept data that multiplex editing with two independent CRISPR/Cas9 ribonucleoproteins (RNPs) targeting genetic loci on chromosomes 11 and 19 robustly maintains long-term in vivo engraftment potential with persistent gene edits at both target sites, with minimally detectable translocations in bone marrow samples 16 weeks after engraftment in NSG mice. The overall viability of dual-edited input CD34+ HSPCs post gene editing was >75% and the proportion of CD34+CD38-CD45RA-CD90+CD49f+ putative long-term hematopoietic stem cells (LT-HSCs) in input cells were similar (1-2%) between unedited and multiplex-edited cells. In vivo engraftment potential of multiplex-edited cells was maintained long-term, with average chimerism of hCD45+ cells in mice transplanted with dual knock-out (KO) HSPCs similar to those transplanted with unedited HSPCs (53.36±11.80% versus 47.45±18.03%, respectively). Dual KO cells also maintained normal differentiation potential as judged by multilineage reconstitution in vivo and in vitro colony forming unit (CFU) assay, indicating the dual RNP delivery process does not lead to any observable impact on the multipotency of the engineered graft. Quantification of on-target editing by multiplexed NGS amplicon sequencing revealed no reduction in total editing between dual KO input and bone marrow cells, indicating the gene modifications in dual engineered cells persist post-engraftment.‡equal contribution, Nipul Patel and Michael A. Pettiglio DisclosuresPettiglio: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Pimentel: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Kapuria: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ghodssi: Vor Biopharma: Current Employment, Current holder of stock options in a privately-held company. Isik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Falla: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Amunugama: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lisle: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Wang: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lydeard: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hazelbaker: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Paik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear.We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology.Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p < 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo.To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p<0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p<0.05), and IFN-γ production was reduced (p<0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E).Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. [Display omitted] DisclosuresBerrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

Preclinical Development of EDIT301, an Autologous Cell Therapy Comprising AsCas12a-RNP Modified Mobilized Peripheral Blood-CD34 + Cells for the Potential Treatment of Transfusion Dependent Beta Thalassemia

Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy.EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny.To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production.In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. DisclosuresSousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Rhesus Macaques As a Preclinical Model Organism for RUNX1-FPD

Germline loss-of-function (LOF) heterozygous mutations in the central hematopoietic transcription factor RUNX1 gene cause the marrow failure/malignancy predisposition syndrome Familial Platelet Disorder with associated Myeloid Malignancies (FPDMM, or FPD). Patients with FPD have defective megakaryocytic development, low platelet counts, and a very high (35-50%) life-long risk of hematological malignancies, particularly MDS/AML. Murine heterozygous gene knockout models do not recapitulate the human phenotype in terms of thrombocytopenia or myeloid leukemia progression. Although gene correction of the RUNX1 mutation in hematopoietic stem and progenitor cells (HSPCs) is being considered as a possible treatment approach, it is unknown whether mutation-corrected HSPCs will have the hoped for advantage over RUNX1 mutant HSCs in vivo, likely necessary to significantly lower leukemia risk. In order to study the relative function of wildtype and RUNX1-mutated HSPCs in vivo in a model with close hematopoietic similarity to humans, we generated a rhesus macaque FPD competitive repopulation model via CRISPR/Cas9 NHEJ editing of the RUNX1 gene versus the AAVS1 safe-harbor control locus. We transplanted mixtures of autologous HSPCs edited at the two loci: 75% RUNX1-edited/25% AAVS1- edited CD34+ HSPCs in animal 1 and 25% RUNX1-edited/75% AAVS1-edited CD34+ HSPCs in animal 2, following conditioning with total body irradiation. Both animals engrafted tri-lineage hematopoiesis promptly following transplantation. However, platelet numbers remained below the normal range long-term in animal 1 receiving a higher ration of RUNX1-edited HSPCS and below counts of macaques receiving HSPCs edited at other loci (Figure 1). Bone marrow morphology at 6 months was normal.To assess the HSPC function of RUNX1 mutant versus AAV1 control and unedited WT cells we tracked RUNX1 and AAVS1-mutated allele frequencies in blood cells over time via deep sequencing (Figure 2). In the infusion products (IP), allele fractions reflected the desired ratios. In both animals, AAVS1-edited cells dominated compared to RUNX1-edited cells. However, in animal 1, RUNX1-mutated cells expanded over time eventually exceeding the ratio in the IP, and in animal 2, levels of RUNX1 and AAVS1-mutated cells were equivalent long-term. Marrow analyzed at 6 months showed heterozygous RUNX1-mutated CFU at levels concordant with mutation frequencies in the blood, but no homozygous RUNX1 mutated CFU, suggesting homozygous LOF is not compatible with long-term HSPC function.In conclusion, we have created pre-clinical model for FPD via CRISPR/Cas editing of HSPCs in rhesus macaques. The lack of a competitive advantage for wildtype or control-locus edited HSPCs over RUNX1 heterozygous-mutated HSPCs long-term in our model suggests that gene correction approaches for FPD will be challenging, particularly to reverse the MDS/AML predisposition phenotype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In This Issue

HomeCirculation ResearchVol. 129, No. 6In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Sep 2021https://doi.org/10.1161/RES.0000000000000503Circulation Research. 2021;129:599is related toSARS-CoV-2 Initiates Programmed Cell Death in PlateletsThe Super-Relaxed State and Length Dependent Activation in Porcine MyocardiumCardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular DystrophyCardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy (p 602)Gene editing reduces heart cell abnormalities caused by dystrophin mutation, say Atmanli et al.Download figureDownload PowerPointDuchenne muscular dystrophy (DMD) affects 1 in 5,000 baby boys and is caused by mutations to the gene for dystrophin—an architectural protein essential for muscle cell integrity. Patients display profound muscle degeneration and weakness, with respiratory and heart muscle dysfunction being major causes of death. With recent improvements in respiratory medicine extending the lives of patients, Atmanli and colleagues focused on heart dysfunction—specifically, whether gene editing could mitigate it. The team created induced pluripotent stem cells (iPSCs) from a DMD patient and his healthy brother and showed that gene editing of the DMD cells enabled their development into normal-looking cardiomyoctes with normal contractile function and calcium handling—equivalent to the healthy control cells. The un-edited DMD cells, by contrast, did not develop normally. For greater clinical relevance, the team edited the DMD cells after cardiomyocyte differentiation, showing this reduced their propensity for arrhythmia compared with that of un-edited cells. Lastly, the team provided evidence to suggest gene editing may improve heart abnormalities in mice with the same mutation. Altogether, the results support the development of gene editing as a treatment for DMD, say the authors.The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium (p 617)Ma et al investigate the mechanics of myofilament length dependent activation.Download figureDownload PowerPointMyofilament length dependent activation (LDA) is the fundamental mechanism coupling the force of the heart’s contraction to its preceding diastolic volume. In other words, it ensures the more the heart fills, the stronger it contracts. While studies of rodent hearts have given insights into LDA mechanics, how it operates in a large mammalian heart is unknown. Using structural and biochemical analyses of pig myocardial fibers, Ma and colleagues show that, compared with small stretches of the fibers (equivalent to small diastolic volumes), long stretches induce greater ATP turnover and greater numbers of cross-bridges between myosin and actin filaments—the contractile machinery proteins. Myosin motors can be either engaged with actin, or in one of two unengaged states—disordered relaxed state, where they are ready to engage, or super relaxed state, where they are essentially switched off. The team showed that as muscle stretch increased, so the amount of super-relaxed myosin motors diminished—with more motors becoming engaged to enable a stronger contraction. Sure enough, when the fibers were treated with a myosin motor inhibitor, these stretch effects were impaired. In revealing the mechanisms of LDA, the work offers a basis for studying cardiomyopathies in which the system has gone awry.SARS-CoV-2 Initiates Programmed Cell Death in Platelets (p 631)In destroying SARS-CoV-2, platelets also destroy themselves, report Koupenova et al.Download figureDownload PowerPointSome patients with COVID-19, especially those with severe disease, exhibit abnormal blood clotting, which can lead to stroke, pulmonary clots or myocardial infarction. Because platelets—small cell-like structures abundant in the blood—play a major role in clotting, researchers have investigated platelet behavior in COVID-19, finding that they can become hyperactivated. What’s not clear, however, is whether the virus acts on platelets directly, or if they become activated due to the inflammatory environment. Koupenova and colleagues have now discovered that SARS-CoV-2 can enter platelets, but that it is quickly fragmented and digested therein to prevent viral replication. Indeed, while some platelets display ACE2 protein—the virus’s entry path—on their surfaces, ACE2 is not needed for entry as platelets more often engulf the viruses into large vacuoles where they are dispatched. This viral disposal comes at a price however. Platelets that gobble up the virus undergo budding and a loss of contents, ultimately succumbing to necroptosis or apoptosis, according to the analyses. And, it is this self-destruction that, if excessive, may contribute to the sort of dysregulated immunity and thrombosis seen in select patients, suggest the authors. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesSARS-CoV-2 Initiates Programmed Cell Death in PlateletsMilka Koupenova, et al. Circulation Research. 2021;129:631-646The Super-Relaxed State and Length Dependent Activation in Porcine MyocardiumWeikang Ma, et al. Circulation Research. 2021;129:617-630Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular DystrophyAyhan Atmanli, et al. Circulation Research. 2021;129:602-616 September 3, 2021Vol 129, Issue 6 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000503 Originally publishedSeptember 2, 2021 PDF download Advertisement

CRISPR-Cas9–mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate–induced inflammation in bovine mammary epithelial cells

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar β-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed thatHBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

SELECTION OF GENOME EDITED HEMATOPOIETIC STEM CELLS PRIOR TO TRANSPLANTATION DECREASES THE ENGRAFTMENT POTENTIAL AND THERAPEUTIC EFFICACY

<h3>Background</h3> Autologous hematopoietic stem cell transplantation using genome edited cells has the potential to become a definitive therapy for lysosomal diseases, including some with neurological involvement. We have previously shown that transplantation with human, genome edited hematopoietic stem and progenitor cells (HSPC) can correct phenotypic and biochemical abnormalities in mice with mucopolysaccharidosis type I (MPSI), a multi-systemic lysosomal disorder caused by alfa-L-iduronidase (IDUA) deficiency and consequent glycosaminoglycan (GAG) storage. Before, we used GFP expression to select edited cells prior to transplantation. To effectively translate this proof-of-concept study to the clinic, we tested a clinically relevant selection marker, the truncated Nerve Growth Factor Receptor (tNGFR), which is not expressed in HSPCs, is not toxic and allows antibody-mediated cell isolation. <h3>Methods</h3> Human HSPCs were CRISPR-edited in the safe harbor CCR5 to express IDUA or IDUA and tNGFR (IDUA-tNGFR). Cells edited with IDUA were transplanted as bulk preparations while IDUA-tNGFR cells were selected with magnetic beads and transplanted as positive (IDUA-tNGFR+) and negative (IDUA-tNGFR-) fractions into NSG-MPSI mice. Phenotypical, molecular and biochemical analyses were performed 18 weeks post-transplant. <h3>Results</h3> Genome editing of IDUA-tNGFR cells was significantly lower than IDUA (14% vs. 31%), possibly due to increased IDUA-tNGFR vector size comparing to IDUA only, reducing the editing efficiency. In transplanted mice, IDUA-tNGFR+ cells engrafted poorly in hematopoietic tissues comparing to IDUA or IDUA-tNGFR- cells. The reduced engraftability and consequent lower therapeutic dose observed with tNGFR-selected cells predicted a worse performance at correcting biochemical features in the MPSI mouse model, with only few mice with detectable levels of the enzyme. Contrarily, mice transplanted with bulk cell preparations had supraphysiological levels of enzyme, with significant biochemical correction even in the brain. <h3>Conclusion</h3> These data suggest that selection of edited HSPCs for transplantation reduces drastically the therapeutic potential of the product, regardless of the selection marker. Therefore, higher therapeutic efficacy is achieved when cells are transplanted as bulk preparations (edited + unedited cells), with significant expression of the therapeutic product also in the brain, constituting a promising therapy for neurodegenerative disorders.

Persistence of CRISPR/Cas9 gene edited hematopoietic stemcells following transplantation: A systematic review andmeta-analysis of preclinical studies.

Gene editing blood‐derived cells is an attractive approach to cure selected monogenic diseases but remains experimental. A systematic search of preclinical controlled studies is needed to determine the persistence of edited cells following reinfusion. All studies identified in our systematic search (to 20 October 2020) examining the use of CRISPR/Cas9 gene editing in blood‐derived cells for transplantation were included. Meta‐analysis was performed to determine the engraftment and persistence of gene edited cells. A total of 3538 preclinical studies were identified with 15 published articles meeting eligibility for meta‐analysis. These in vivo animal studies examined editing of hemoglobin to correct sickle cell disease (eight studies), inducing resistance to acquired immunodeficiency syndrome (two studies), and six other monogenic disorders (single studies). CRISPR‐Cas9 edited hematopoietic stem and progenitor cells demonstrated equivalent early engraftment compared to controls in meta‐analysis but persistence of gene‐edited cells was reduced at later time points and in secondary transplant recipients. Subgroup analysis in studies targeting the hemoglobin gene revealed a significant reduction in the persistence of gene‐edited cells whether homology‐directed repair or nonhomologous end‐joining were used. No adverse side effects were reported. Significant heterogeneity in study design and outcome reporting was observed and the potential for bias was identified in all studies. CRISPR‐Cas9 gene edited cells engraft similarly to unedited hematopoietic cells. Persistence of gene edited cells, however, remains a challenge and improved methods of targeting hematopoietic stem cells are needed. Reducing heterogeneity and potential risk of bias will hasten the development of informative clinical trials.

Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis

The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Adenosine Base Editing of γ-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T&amp;gt;C, -175 T&amp;gt;C and -113 A&amp;gt;G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were &amp;lt;2% at each of the three sites, reflecting a low rate of DSBs. Fetal hemoglobin levels in erythroid cells generated in vitro from A base-edited CD34+ HSPCs were 26±4% (-198 T&amp;gt;C), 60±10% (-175 T&amp;gt;C), and 42±7% (-113 A&amp;gt;G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T&amp;gt;C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P&amp;gt;0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T&amp;gt;C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents &amp; Royalties; Editas Medicine: Consultancy, Patents &amp; Royalties; Beam Therapeutics: Consultancy, Patents &amp; Royalties; Prime Medicine: Consultancy, Patents &amp; Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

CRISPR Mediated ELANE Single-Allele Knock-out Restores Proliferation and Myeloid Differentiation of Neutropenia Patient Derived BM HSCs

Background: Mutations in ELANE are a cause of cyclic and severe congenital neutropenia (SCN), predominantly autosomal dominant disorders. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease. Neutropenia occurs because mutant NE impairs survival and maturation of myeloid cells. More than 130 disease causing mutations in ELANE have been identified and cell permeable inhibitors of NE can correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). CRISPR/Cas9 knock-out (KO) of ELANE in patients' hematopoietic stem cells (HSCs) corrects myeloid cell differentiation, (Nasri et al Haematologica 2020:105:598), but this approach targets both alleles, eliminates normal NE and may impair innate immunity. Hypothesis: Selective, single allele editing is a preferred strategy for correcting ELANE associated neutropenia and other autosomal dominant disorders. Methods: Emendo Biotherapeutics developed CRISPR-associated allele specific KO based on single nucleotide polymorphisms (SNPs) located in the vicinity of ELANE. Three SNPs cover ~80% of the population. In the current study, editing of the mutated allele was achieved by targeting SNP rs1683564 located downstream to the 3'UTR of ELANE and mediating a biallelic break in intron 4, with excision of the 3'UTR, destabilized the mutated allele transcript. For editing we utilized RNPs assembled from Emendo's proprietary nuclease, OMNI-50, a guide targeting the SNP and a guide targeting a region in intron 4. OMNI-50 has low off-target activity and high allele specific editing when targeting either the reference or the alternative form of the SNPs. The efficacy of this composition was tested on normal and SCN patient-derived HSCs harboring the relevant SNP. (Figure 1) A patient's bone marrow CD34+ cells with the S126L mutation and rs1683564 were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies), purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and purity verified by FACS analysis using CD34 and CD45 antibodies. Purified CD34+ cells were cryopreserved using serum free CryoStor® CS10 media (Stemcell Technologies) and stored in Liquid Nitrogen Vapor Phase. Normal human blood CD34+ progenitor cells, harboring rs1683564 were similarly treated as the control. Allele specific excision was determined by ddPCR. To determine excision efficiency for the patient and control, we amplified two regions in ELANE, Exon 1 and Exon 5 (excised region), using two different probes labeled with FAM and HEX, respectively. We used OMNI-50 nuclease (Emendo Bio, Ness Ziona, Israel), electroporation (Lonza 4D nucleofector) and StemCell media (Stemcell Technologies, Vancouver, BC) to modify, grow and differentiate the CD34+ cells. We assessed myeloid cell proliferation and differentiation using daily cell counts, cytospins stained with Diff-Quik, and CD14, CD66b, CD15, CD16, CD11b labeling and flow cytometry. Results: Unedited patient cells demonstrated significant abnormalities in proliferation and differentiation. Cytospins from unedited patient CD34+ cells at 14 days showed block of myeloid differentiation and 4-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Single allele ELANE KO significantly improved these cellular abnormalities: total cell proliferation increased 41% and CD14/CD66b and CD15/CD11b positive cells increased by 107% and 65.5%, respectively. Studies are in progress for additional patients harboring different ELANE mutations. Conclusions: Single allele KO may resolve concerns about unwanted effects of total elimination of the normal gene products with gene editing for autosomal dominant diseases and be an effective strategy for treating ELANE associate neutropenia. Disclosures Sabo: Emendo BioTherapeutics: Research Funding. Makaryan:Emendo BioTherapeutics: Research Funding. Poulsen:Emendo BioTherapeutics: Research Funding. Povodovski:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dicken:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Herman:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Emmanuel:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dale:X4 Pharmaceuticals: Research Funding; Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Honoraria.

Robust Pre-Clinical Results and Large-Scale Manufacturing Process for Edit-301: An Autologous Cell Therapy for the Potential Treatment of SCD

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. As fetal hemoglobin (HbF) has been shown to be protective against clinical manifestation of SCD, we are developing EDIT-301, an autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a ribonucleoprotein (RNP) to promote HbF expression to treat SCD. Fetal hemoglobin induction for EDIT-301 is achieved by disrupting the HBG1 and HBG2 promoter distal CCAAT-box region where naturally occurring mutations are found to be associated with elevated HbF expression. Cas12a was selected over Cas9 due to the more productive and sustainable (NHEJ derived) indel profile, as well as high specificity. Using Cas12a RNP, on-target editing of ~90% was achieved in mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from both healthy and SCD donors at research scale with no detectable off targets. Editing of CD34+ cells led to an average of 43% and 54% of HbF expression in the erythroid progeny of normal donor and SCD donor cells respectively in a pancellular fashion (~93% population). The robust HbF induction in SCD red blood cells (RBCs) resulted in significant phenotypic and functional improvement including reduced sickling and increased deformability under hypoxia ex vivo. Using a microfluidic assay that replicated blood flow in microvasculature under varying oxygen conditions, SCD RBCs derived from RNP electroporated CD34+ cells showed improved rheological behavior. The rheology improvement under hypoxia was strongly correlated with the increased levels of HbF in each sample. Infusion of the modified CD34+ cells from normal donors into NBSGW mice resulted in long-term multi lineage and polyclonal reconstitution. Editing levels at 16 weeks post infusion were &amp;gt; 90% in all human lineages tested, demonstrating the efficient editing of SCID-repopulating hematopoietic stem cells (HSCs). Consistent with the high editing levels, human erythroid cells from the bone marrow of mice that received Cas12a-RNP treated cells demonstrated pancellular (~90% F+ RBCs) HbF expression averaging 40-50% of total hemoglobin compared to ~5% HbF observed in the control group. We have developed a consistent large-scale process using functionally closed, semi-automated systems suitable for use in clinical manufacturing. We have shown robust editing of normal donor CD34+ cells and SCID-repopulating HSCs with the clinical scale process. Editing levels of &amp;gt;90% were detected after long term engraftment in mice. In summary, we have demonstrated successful on-target editing of mPB CD34+ cells derived from both normal and SCD donors using a Cas12a RNP, which coincided with robust HbF induction and a phenotypic reduction of sickling in the SCD erythroid progeny, as well as improved rheological behavior. Editing of the HBG1 and HBG2 promoters using this RNP was highly specific with no measurable off-target. In vivo, cells from normal donors readily engrafted and reconstituted all blood cell lineages at levels comparable to unedited cells. Finally, a robust large-scale manufacturing process has been developed to supply material for the clinical setting. Based on these results, we are completing the activities required to assess EDIT-301 in the clinic as treatment for SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Heath:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Sousa:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Janoudi:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. An:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Albright:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Teixeira:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Monesmith:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Chang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company.

Functional analysis of bovine interleukin-10 receptor alpha in response to Mycobacterium avium subsp. paratuberculosis lysate using CRISPR/Cas9

BackgroundThe interleukin-10 receptor alpha (IL10RA) gene codes for the alpha chain of the IL-10 receptor which binds the cytokine IL-10. IL-10 is an anti-inflammatory cytokine with immunoregulatory function during the pathogenesis of many inflammatory disorders in livestock, including Johne’s disease (JD). JD is a chronic enteritis in cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is responsible for significant economic losses to the dairy industry. Several candidate genes including IL10RA have been found to be associated with JD. The aim of this study was to better understand the functional significance of IL10RA in the context of immune stimulation with MAP cell wall lysate.ResultsAn IL10RA knock out (KO) bovine mammary epithelial cell (MAC-T) line was generated using the CRISPR/cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) gene editing system. These IL10RA KO cells were stimulated with the immune stimulant MAP lysate +/− IL-10, or with LPS as a positive control. In comparison to unedited cells, relative quantification of immune-related genes after stimulation revealed that knocking out IL10RA resulted in upregulation of pro-inflammatory cytokine gene expression (TNFA, IL1A, IL1B and IL6) and downregulation of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of pro-inflammatory cytokine signaling. At the protein level knocking out IL10RA also resulted in upregulation of inflammatory cytokines - TNF-α and IL-6 and chemokines - IL-8, CCL2 and CCL4, relative to unedited cells.ConclusionsThe findings of this study illustrate the broad and significant effects of knocking out the IL10RA gene in enhancing pro-inflammatory cytokine expression and further support the immunoregulatory role of IL10RA in eliciting an anti-inflammatory response as well as its potential functional involvement during the immune response associated with JD.

Genome editing of CCR5 by AsCpf1 renders CD4+T cells resistance to HIV-1 infection

BackgroundThe chemokine receptor CCR5 is one of the co-receptor of HIV-1 infection. People with homozygous CCR5Δ32 deletion resist HIV-1 infection, which makes the CCR5 an important target for HIV-1 gene therapy. Although the CRISPR/Cas9 has ever been used for HIV-1 study, the newly developed CRISPR/AsCpf1 has never been utilized in HIV-1 co-receptor disruption. The CRISPR/Cpf1 system shows many advantages over CRISPR/Cas9, such as lower off-target, small size of nuclease, easy sgRNA design for multiplex gene editing, etc. Therefore, the CRISPR/Cpf1 mediated gene editing will confer a more specific and safe strategy in HIV-1 co-receptor disruption.ResultsHere, we demonstrated that CRISPR/AsCpf1 could ablate the main co-receptor of HIV-1 infection-CCR5 efficiently with two screened sgRNAs via different delivery strategies (lentivirus, adenovirus). The edited cells resisted R5-tropic HIV-1 infection but not X4-tropic HIV-1 infection compared with the control group in different cell types of HIV-1 study (TZM.bl, SupT1-R5, Primary CD4+T cells). Meanwhile, the edited cells exhibited selective advantage over unedited cells while under the pressure of R5-tropic HIV-1. Furthermore, we clarified that the predicted off-target sites of selected sgRNAs were very limited, which is much less than regular using sgRNAs for CRISPR/Cas9, and no evident off-target was observed. We also showed that the disruption of CCR5 by CRISPR/AsCpf1 took no effects on cell proliferation and apoptosis.ConclusionsOur study provides a basis for a possible application of CCR5-targeting gene editing by CRISPR/AsCpf1 with high specific sgRNAs against HIV-1 infection.

Immunity to Cas9 as an Obstacle to Persistent Genome Editing

Immunity to Cas9 as an Obstacle to Persistent Genome Editing