Nuclease Platforms Research Articles

Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.

Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.

Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP.

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.

The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements.

The common IVSI-110 (G>A) β-thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G>A) transgenic cells and the disruption events were compared with published observations in primary CD34+ cells. DARE achieved the functional correction of β-globin expression equally through the removal of causative mutations and through the removal of context sequences, with disruption events and the restriction of indel events close to the cut site closely resembling those seen in primary cells. Correlation of DNA-, RNA-, and protein-level findings then allowed the extrapolation of findings to other mutations by in silico analyses for potential repair based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, Cas12a, and transcription activator-like effector nuclease (TALEN) platforms. The high efficiency of DARE and unexpected freedom of target design render the approach potentially suitable for 14 known thalassemia mutations besides IVSI-110(G>A) and put it forward for several prominent mutations causing other inherited diseases. The application of DARE, therefore, has a wide scope for sustainable personalized advanced therapy medicinal product development for thalassemia and beyond.

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

Genome editing for blood disorders: state of the art and recent advances.

In recent years, tremendous advances have been made in the use of gene editing to precisely engineer the genome. This technology relies on the activity of a wide range of nuclease platforms — such as zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR–Cas system — that can cleave and repair specific DNA regions, providing a unique and flexible tool to study gene function and correct disease-causing mutations. Preclinical studies using gene editing to tackle genetic and infectious diseases have highlighted the therapeutic potential of this technology. This review summarizes the progresses made towards the development of gene editing tools for the treatment of haematological disorders and the hurdles that need to be overcome to achieve clinical success.

Enhanced Cas12a editing in mammalian cells and zebrafish.

Type V CRISPR–Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.

Chemistry, manufacturing and controls for gene modified hematopoietic stem cells

Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e.g., Zinc finger, TALEN and Crispr/Cas9) are being applied to the treatment of multiple genetic and immunodeficiency disorders. Herein, the current state of the art in manufacturing and critical assays that are required for ex vivo manipulation of stem cells are addressed. Important quality control and safety assays that need to be planned early in the process development phase of these products for regulatory approval are also highlighted.

Engineering of CRISPR-Cas12b for human genome editing

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient's own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model.The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion.We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing.These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases.Figure 1:Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A). DisclosuresNegre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair:RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

Multiplex Human T Cell Engineering without Double-Strand Break Induction Using the Cas9 Base Editor System

Chimeric antigen-receptor (CAR) engineered T cells have mediated impressive outcomes in a subset of hematological malignancies, yet this therapy remains highly personalized and largely ineffective against more widespread epithelial and solid tumors. Cellular manufacturing processes tailored to individual patients, and an inability to overcome the complex and immunosuppressive properties of the solid tumor microenvironment remain formidable challenges in the effort to achieve widespread application of adoptive cell therapies for cancer. Genome editing strategies using targeted nuclease platforms are being developed to overcome these limitations, and several are already entering clinical application. Multiplex gene editing strategies to develop off-the-shelf cellular therapies against non-hematological malignancies are of particularly high interest but remain limited by concerns surrounding off-target nuclease activity and chromosomal translocations induced by simultaneous double-strand break (DSB) induction at multiple genomic loci. The risk of genotoxic side-effects is further amplified when combining multiplex DSB induction with randomly integrating platforms for antigen-specific receptor delivery.An ideal strategy would allow for multi-gene disruption and targeted integration of antigen-specific receptors without introducing multiple genomic DSB. To this end, we evaluated the application of third- and fourth-generation Cas9 base-editor technologies for gene disruption in primary human T cells. Through systematic reagent and dose optimization efforts we achieved highly efficient, targeted cytosine to thymine (C > T) base conversion (Figure 1A) and consequent protein knockout (Figure 1B) at multiple therapeutically relevant loci including TRAC (KO = 80.8%, SD = 10.7%), PD1 (79%, SD = 4%), and B2M (80%, SD = 3%). We observed that fourth generation base editor (BE4) achieved consistently higher C > T conversion rates with reduced non-canonical editing (i.e. C>A, G) overall compared to third generation (BE3). Targeted disruption of splice acceptor (SA) and splice donor (SD) sites resulted in higher frequency of protein knockout versus induction of premature stop codons at all loci examined.Importantly, while multiplex editing of these loci using Cas9 nuclease resulted in detectable translocations between all targeted sites, these translocations were largely eliminated using BE4 (Figure 1C). Finally, we exploited both the single strand nickase function of BE4 and orthogonal Cas enzymes in conjunction with rAAV delivery to achieve targeted integration of a gene expression cassette at the AAVS1 safe harbor locus (Figure 2A,B). Targeted integration using an orthogonal Cas9 system (SaCas9) in conjunction with multiplex base editing achieved simultaneous gene knockin and gene knockout (Figure 2C). Engineered T cells expand to clinically relevant numbers and maintain cytokine polyfunctionality and the ability to kill target cells when equipped with a chimeric antigen receptor. Collectively, we demonstrate that Cas9 base-editor technology can be utilized to mediate efficient, multiplex gene disruption and targeted gene integration in primary human T cells without associated translocations. This streamlined approach to genome engineering may be broadly applied for the development of safe and effective cell therapies. [Display omitted] DisclosuresWebber:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Moriarity:B-MoGen Biotechnologies: Employment, Equity Ownership; BEAM Therapeutics: Consultancy.

Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells

Chimeric antigen receptor (CAR) T cell therapies continue to show excellent outcomes in hematological cancers. Achieving success in additional tumor indications, however, will likely require modulating inhibitory pathways that limit CAR T cell potency. We developed a megaTAL nuclease targeting the gene encoding Casitas B-lineage lymphoma proto-oncogene-b (CBLB), a ubiquitin ligase that serves as an intracellular checkpoint that negatively regulates T cell activation. The megaTAL nuclease platform has been previously shown to drive highly efficient genome editing in primary T cells. Electroporation of primary T cells with mRNA encoding the CBLB megaTAL resulted in >90% indels at the target locus and a concomitant reduction of Cbl-b protein levels. Specificity characterization studies revealed three detectable non-exonic off-target sites with near negligible indel frequencies. We next assessed the functional impact of CBLB disruption in CAR T cells engineered to target the epidermal growth factor receptor (EGFR). When co-cultured with EGFR+ target cells, CAR T cells with megaTAL-mediated CBLB gene knockout had a 2-fold increase in pro-inflammatory cytokine production compared with mock-treated CAR T cells. We developed an A549 tumor xenograft model to test the activity of CBLB megaTAL-treated CAR T cells in vivo. While mock-treated CAR T cells had a transient impact on tumor growth, we observed complete and durable tumor elimination in mice infused with the CBLB megaTAL-treated CAR T cells. Improved responses in the megaTAL treated animals were particularly pronounced at lower CAR T cell doses, suggesting that CBLB knockout enhances the potency of CAR T cells. In summary, the CBLB megaTAL is a highly efficient and specific gene editing nuclease that enhances CAR T cell anti-tumor responses in vitro and in vivo, and thus could potentially improve the efficacy of CAR T therapy. DisclosuresHooper:bluebird bio: Employment, Equity Ownership. Havens:bluebird bio: Employment, Equity Ownership. Krostag:bluebird bio: Employment, Equity Ownership. Magee:bluebird bio: Employment, Equity Ownership. Martin:bluebird bio: Employment, Equity Ownership. Gupta:bluebird bio: Employment, Equity Ownership. Smurnyy:bluebird bio: Employment, Equity Ownership. Pechilis:bluebird bio: Employment, Equity Ownership. Rode:bluebird bio: Employment, Equity Ownership. Chavkin:bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership.

GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases

BackgroundGenome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed.ResultsHere, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction algorithm permitting the identification of genomic sequences with unexpected cleavage activity.ConclusionThe GUIDEseq package enables analysis of GUIDE-data from various nuclease platforms for any species with a defined genomic sequence. This software package has been used successfully to analyze several GUIDE-seq datasets. The software, source code and documentation are freely available at http://www.bioconductor.org/packages/release/bioc/html/GUIDEseq.html.

Abstract B106: Substantial enrichment of HIV- resistant CD4 T cells by combining the targeted integration of a resistance-cassette at the CCR5 locus with chemoselection

Abstract Engineered nucleases can be used to induce double stranded breaks at specific sites in the genome to facilitate therapeutic gene disruption or gene insertion. Gene insertion strategies may have a significant advantage over traditional lentiviral gene therapy since we have much greater control over transgene integration site, copy number, and expression. However, a major limitation in the clinical application of gene insertion technologies is the low efficiency of homology driven repair (HDR) achieved in hematopoietic cells. Here, we report the knock-in of a selection marker, which allows us to enrich for modified CD4 T cells, and to achieve clinically relevant levels of gene edited cell populations in the disease-context of HIV. Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4 T cells and represents an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining a high level of CCR5 gene disruption with preferential selection of gene-modified cells using a megaTAL nuclease. The CCR5-targeting megaTAL, combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site-specific DNA cleavage. This nuclease produces highly efficient CCR5 disruption in primary human CD4+ T cells in vitro (70-90%). To test the protective effects of gene disruption, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Next, to overcome the decline of HIV-resistant cells, observed in the clinical trials to date, we proposed to couple CCR5 gene-disruption with gene insertion of mutant human dihydrofolate reductase (mDHFR). The mDHFR chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We first tested our experimental approach by transducing cells with a lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.2uM. This approach resulted in a six-fold enrichment of gene modified primary CD4+ T cells ex vivo. For gene insertion, combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we merged the megaTAL nuclease and AAV gene-delivery treatments to integrate mDHFR into the CCR5 locus, thus producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.8kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in 0.1uM MTX compared to untreated controls ex vivo. Finally, we are assessing the therapeutic potential of our approach in NSG mice. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus. Eventually this system could be used to select for modified cells in patients using previously established protocols for MTX administration in the clinic. Citation Format: Biswajit Paul, Guillermo Romano Ibarra, David J. Rawlings, Hans-Peter Kiem. Substantial enrichment of HIV- resistant CD4 T cells by combining the targeted integration of a resistance-cassette at the CCR5 locus with chemoselection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B106.

The emerging role of viral vectors as vehicles for DMD gene editing.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding DMD gene. The DMD gene, spanning over 2.4 megabases along the short arm of the X chromosome (Xp21.2), is the largest genetic locus known in the human genome. The size of DMD, combined with the complexity of the DMD phenotype and the extent of the affected tissues, begs for the development of novel, ideally complementary, therapeutic approaches. Genome editing based on the delivery of sequence-specific programmable nucleases into dystrophin-defective cells has recently enriched the portfolio of potential therapies under investigation. Experiments involving different programmable nuclease platforms and target cell types have established that the application of genome-editing principles to the targeted manipulation of defective DMD loci can result in the rescue of dystrophin protein synthesis in gene-edited cells. Looking towards translation into the clinic, these proof-of-principle experiments have been swiftly followed by the conversion of well-established viral vector systems into delivery agents for DMD editing. These gene-editing tools consist of zinc-finger nucleases (ZFNs), engineered homing endoculeases (HEs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs) based on clustered, regularly interspaced, short palindromic repeats (CRISPR)–Cas9 systems. Here, we succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies.

43. CRISPR/Cas9 and rAAV6-Mediated Targeted Integration at the CCR5 Locus in Hematopoietic Stem and Progenitor Cells

CRISPR/Cas9-mediated genome editing relies on guide RNAs to direct site-specific DNA cleavage mediated by the Cas9 endonuclease. In this study, we identified a highly potent single guide RNA (sgRNA) targeting exon 3 of CCR5. This sgRNA was chemically synthesized with three modified nucleotides at each terminus with 2′O-Methyl and phosphorothioate modifications, and electroporated into cells either with Cas9 mRNA or complexed with Cas9 protein (RNP). Using Tracking of Indels by DEcomposition (TIDE) to quantify insertions and deletions (INDELs), we observe up to 80% INDELs in CD34+ hematopoietic stem and progenitor cells (HSPCs).To achieve targeted integration by homology-directed repair (HDR), we produced rAAV6 vectors carrying a GFP expression cassette flanked by CCR5 homology arms. Electroporation with Cas9 RNP followed by rAAV6 transduction led to targeted integration in up to 30% of the cells. Interestingly, we observed that cells with targeted integration expressed GFP at fluorescence intensities more than 10-fold higher than from episomal AAV vectors. This allowed us to sort targeted cells as early as four days after nucleofection and transduction. Upon fluorescence-activated cell sorting and culture of this population, >99% of cells remained GFP+ 20 days post sort. In a methylcellulose-based colony-forming unit (CFU) assay, we identified multipotent and lineage-committed progenitor cells in this population, and PCR of gDNA extracted from colonies confirmed targeted integration at CCR5 in at least 98% of cells. Phenotypic characterization of this targeted population confirmed the presence of CD34+ CD38− CD90+ CD45RA− cells, indicating genome editing of hematopoietic stem cells. We transplanted edited cells into immunodeficient NSG mice and analyzed the bone marrow 8 weeks post-transplant. In mice transplanted with cells that were not enriched for targeted integration, we found 0.1-1.9% GFP+ cells among the engrafted human cells. This was a significant decline compared to the 12-13% GFP+ cells in the input cell population following culture, which is a phenomenon consistent with findings reported by other groups using different nuclease platforms. In contrast, when transplanting cells enriched for targeted integration, we found that 75% of the engrafted human cells were GFP+, confirming the presence of cells with long-term engraftment potential in the enriched population.In conclusion, we have found that the combination of CRISPR/Cas9 and rAAV6 is an effective platform for HDR-mediated targeted integration of a transgene into the CCR5 locus. Furthermore, the GFP MFI shift observed when episomal rAAV6 vectors are integrated into the chromosome following HDR enables early isolation of a population highly enriched for targeted integration at this locus. Since CCR5 is considered a ‘safe harbor’ for targeted insertion of a gene, this approach might find general use in therapeutic genome editing. Additionally, since CCR5 is an important co-receptor during HIV infection, our findings may be used to generate immune cells resistant to HIV infection.

44. Novel Combination of megaTAL Nuclease-Driven Genome Engineering with a Drug Selection Cassette Increases Efficiency of HIV Gene Therapy

Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4+ T cells and represent an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining high level of CCR5 gene disruption with preferential selection of gene modified cells. The CCR5-targeting megaTAL combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site specific DNA cleavage. This nuclease produces highly efficient CCR5 targeting in primary human CD4+ T cells in vitro (70-90% disruption). To test the protective effects of megaTAL treatment, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Based on the decline of CCR5 modified cells in the clinical trials to date, we hypothesized that we could improve maintenance of HIV resistant cells by expanding them either ex vivo or in vivo. We propose that coupling megaTAL nuclease treatment with drug selection will help us achieve therapeutically relevant levels of HIV-protected cells by enabling efficient selection only of CCR5-modified T-cells. The mutant human dihydrofolate reductase (mDHFR) chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We tested our experimental approach by transducing cells with lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.02uM. This approach resulted in a six fold enrichment of gene modified primary CD4+ T cells ex vivo. Previously we have shown that combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we combined megaTAL/AAV treatment to integrate the mutant DHFR into the CCR5 locus, producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.6kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in MTX compared to untreated controls ex vivo. Next, we have transplanted NSG mice with 1×106 gene modified cells/mouse to assess the therapeutic potential of our approach. Mice that engraft effectively will be treated with daily injections of 0, 0.5 and 2 mg/kg of MTX to monitor preferential selection and enrichment of our target cell population. Subsequent studies will assess the long term control of viremia in these mice following HIV challenge. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Soybean (Glycine max) Mutant and Germplasm Resources: Current Status and Future Prospects.

Genetic bottlenecks during domestication and modern breeding limited the genetic diversity of soybean (Glycine max (L.) Merr.). Therefore, expanding and diversifying soybean genetic resources is a major priority for the research community. These resources, consisting of natural and induced genetic variants, are valuable tools for improving soybean and furthering soybean biological knowledge. During the twentieth century, researchers gathered a wealth of genetic variation in the forms of landraces, Glycine soja accessions, Glycine tertiary germplasm, and the U.S. Department of Agriculture (USDA) Type and Isoline Collections. During the twenty-first century, soybean researchers have added several new genetic and genomic resources. These include the reference genome sequence, genotype data for the USDA soybean germplasm collection, next-generation mapping populations, new irradiation and transposon-based mutagenesis populations, and designer nuclease platforms for genome engineering. This paper briefly surveys the publicly accessible soybean genetic resources currently available or in development and provides recommendations for developing such genetic resources in the future. © 2016 by John Wiley & Sons, Inc.

Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing.

Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.