Engineered Zinc Finger Nucleases Research Articles

A Potential Therapy for Beta-Thalassemia (ST-400) and Sickle Cell Disease (BIVV003)

▪Background: Beta-thalassemia and sickle cell disease are genetic disorders caused by mutations in the beta-globin gene which lead to significant anemia and serious medical complications. Increases in fetal hemoglobin (HbF) have been linked to improved clinical outcomes in patients with beta-thalassemia (BT) and sickle cell disease (SCD).Methods: We have developed engineered zinc finger nucleases (ZFNs) that precisely cleave and disrupt the erythroid enhancer of the BCL11A gene, which substantially boosts HbF production in erythroid progeny of genome-edited CD34+ hematopoietic stem/progenitor cells (HSPCs). The autologous modified HSPC drug product are named ST-400 (BT) and BIVV003 (SCD).Results: We demonstrate that ST-400 / BIVV003 can be manufactured by reproducible, high-level, ZFN-driven modification in peripheral blood mobilized HSPCs at clinical production scale (>108 cells). The drug product is made in a GMP-compliant setting using a clinical-grade electroporation device to deliver the ZFN mRNAs ex vivo . Unbiased specificity studies of ST-400 / BIVV003 demonstrated an exquisite amount of specificity, with high levels of on-target modification (~80%). Erythroid colony genotyping in enhancer targeted cells, showed bi-allelic modification of the BCL11A erythroid enhancer in >50% of HSPCs, resulting in >4-fold higher levels of gamma globin mRNA and protein compared to controls. Similarly, we observed high levels of modification in research-scale preparations of HSPCs from patients with beta-thalassemia. Injection of ST-400 / BIVV003 into immune-deficient mice resulted in robust long-term (19 week) engraftment. Targeted gene modification was maintained through multi-lineage differentiation in the bone marrow and peripheral blood.Conclusions: These results support further clinical development of ST-400 / BIVV003 as a potential therapy for beta-thalassemia and sickle cell disease. DisclosuresNo relevant conflicts of interest to declare.

Non-viral Delivery of Zinc Finger Nuclease mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets

It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable>90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of invivo genome editing and can potentially offer a new treatment modality for a variety of diseases.

Engineered zinc-finger nuclease to generate site-directed modification in the KLF1 gene for fetal hemoglobin induction.

Elevation of hemoglobin F (HbF) ameliorates symptoms of β-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to β hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in β-thalassemia patients.

185 - A Potential Therapy for Beta-Thalassemia (ST-400) and Sickle Cell Disease (BIVV003)

Background: Beta-thalassemia and sickle cell disease are genetic disorders caused by mutations in the beta-globin gene which lead to significant anemia and serious medical complications. Increases in fetal hemoglobin (HbF) have been linked to improved clinical outcomes in patients with beta-thalassemia (BT) and sickle cell disease (SCD). Methods: We have developed engineered zinc finger nucleases (ZFNs) that precisely cleave and disrupt the erythroid enhancer of the BCL11A gene, which substantially boosts HbF production in erythroid progeny of genome-edited CD34 + hematopoietic stem/progenitor cells (HSPCs). The autologous modified HSPC drug product are named ST-400 (BT) and BIVV003 (SCD). Results: We demonstrate that ST-400 / BIVV003 can be manufactured by reproducible, high-level, ZFN-driven modification in peripheral blood mobilized HSPCs at clinical production scale (>10 8 cells). The drug product is made in a GMP-compliant setting using a clinical-grade electroporation device to deliver the ZFN mRNAs ex vivo . Unbiased specificity studies of ST-400 / BIVV003 demonstrated an exquisite amount of specificity, with high levels of on-target modification (~80%). Erythroid colony genotyping in enhancer targeted cells, showed bi-allelic modification of the BCL11A erythroid enhancer in >50% of HSPCs, resulting in >4-fold higher levels of gamma globin mRNA and protein compared to controls. Similarly, we observed high levels of modification in research-scale preparations of HSPCs from patients with beta-thalassemia. Injection of ST-400 / BIVV003 into immune-deficient mice resulted in robust long-term (19 week) engraftment. Targeted gene modification was maintained through multi-lineage differentiation in the bone marrow and peripheral blood. Conclusions: These results support further clinical development of ST-400 / BIVV003 as a potential therapy for beta-thalassemia and sickle cell disease. Disclosures No relevant conflicts of interest to declare.

Clinical-Scale Genome Editing of the Human BCL11A Erythroid Enhancer for Treatment of the Hemoglobinopathies

We describe here a fundamentally novel way to develop a disease therapeutic: combining genome-wide association studies (GWAS) with targeted genome editing to create, in a clinically compliant setting, a disease-ameliorating genotype in the patient's own cells. In β-thalassemia, elevated levels of fetal hemoglobin (HbF) lessen or eliminate disease symptoms, thus making a reversal of HbF silencing in patients an appealing therapeutic strategy. Loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, elevate HbF; rare individuals carrying a monoallelic knockout of BCL11A exhibit no known hematologic abnormality and up to 30% circulating HbF. We previously reported de novo knockout of BCL11A using targeted genome editing with engineered zinc finger nucleases (ZFNs) yielding up to 40% HbF in erythroid progeny of edited human CD34 cells in vitro. We now find that the targeted ablation of a single, specific GATAA motif in the BCL11A intronic enhancer does not affect in vitro erythroid differentiation, but reproducibly (n=6) activates fetal globin transcription in erythroid progeny of modified CD34 cells; importantly, at similar levels of on-target marking in CD34+ cells, these effects on fetal globin mRNA are comparable to those resulting from ZFN-driven coding knockout of BCL11A itself. We demonstrate reproducible (n=8), high-efficiency (up to 82%; average, 69%) ZFN-driven marking at the enhancer in peripheral blood mobilized human CD34 cells at clinical production scale (>1e8 cells) in a GMP-compliant setting for which we use a clinical-grade electroporation device to deliver nuclease-encoding transcribed mRNA ex vivo. Using erythroid colony assay genotyping we find that up to 70% of the cells in the resulting population are biallelically modified at the target locus, while ~10% remain wild-type, and find comparably high levels of marking in research-scale preparations of CD34 cells from patients with β-thalassemia. We observe robust long-term (18-24 week) engraftment and multilineage differentiation of genome-edited cells in immunodeficient mice, similar to control cells, and equivalent modification at the targeted enhancer locus at all timepoints in both differentiated (CD19+, CD3+, CD33+) and more primitive progenitor (CD34+CD38low) cells of human origin purified from bone marrow of long-term-engrafted animals. Our findings support clinical development of enhancer editing as a treatment of the β hemoglobinopathies with autologous hematopoietic stem cell transplant. DisclosuresUrnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Vierstra:University of Washington: Patents & Royalties: Patent applications have been filed based on this work. Chang:Biogen: Employment, Equity Ownership. Zhou:Sangamo BioSciences: Employment, Equity Ownership. Mich:Sangamo BioSciences: Employment, Equity Ownership. Adrian:Cellerant Therapeutics: Equity Ownership; Sangamo BioSciences: Employment, Equity Ownership. Fox:Sangamo BioSciences: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Craig:Sangamo BioSciences: Employment, Equity Ownership. Rebar:Sangamo BioSciences: Employment. Stamatoyannopoulos:University of Washington: Patents & Royalties: Patent applications have been filed based on this work.. Jiang:Biogen: Employment, Equity Ownership.

Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease

Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms. Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs). However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients. Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs).We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation. The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability. Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition. Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher.The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model. At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells. However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN. The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage. In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence. Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD. DisclosuresTan:Biogen: Employment, Equity Ownership. Chang:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Zhou:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

Recent developments and clinical studies utilizing engineered zinc finger nuclease technology.

Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using homologous recombination, the efficiency is extremely low. Zinc finger nucleases (ZFNs) are custom-designed artificial nucleases that make double-strand breaks at specific sequences, enabling efficient targeted genetic modifications such as corrections, additions, gene knockouts and structural variations. ZFNs are composed of two domains: (i) a DNA-binding domain comprised of zinc finger modules and (ii) the FokI nuclease domain that cleaves the DNA strand. Over 17 years after ZFNs were initially developed, a number of improvements have been made. Here, we will review the developments and future perspectives of ZFN technology. For example, ZFN activity and specificity have been significantly enhanced by modifying the DNA-binding domain and FokI cleavage domain. Advances in culture methods, such as the application of a cold shock and the use of small molecules that affect ZFN stability, have also increased ZFN activity. Furthermore, ZFN-induced mutant cells can be enriched using episomal surrogate reporters. Additionally, we discuss several ongoing clinical studies that are based on ZFN-mediated genome editing in humans. These breakthroughs have substantially facilitated the use of ZFNs in research, medicine and biotechnology.

479. ZFN-Mediated In Vivo Genome Editing Results in Supraphysiological Levels of Lysosomal Enzymes Deficient in Hunter and Hurler Syndrome and Gaucher Disease

Lysosomal storage diseases (LSDs) represent a group of inherited metabolic disorders associated with mutations in genes encoding lysosomal enzymes leading to systemic accumulation of toxic storage materials. Manifestations include organomegaly, skeletal dysplasias, cardiopulmonary obstruction, and severe neurologic impairment, often leading to death by age 10. Current LSD therapies include enzyme replacement therapy, substrate reduction therapy and hematopoietic stem cell transplant (HSCT). However, all of these therapies are expensive, incompletely effective, and HSCT is associated with significant risk of morbidity and mortality. Due to the unmet need genome editing strategies are proposed to permanently modify patient cells by genetically complementing the LSD defect. This is achieved by utilizing engineered zinc finger nucleases (ZFNs) to introduce a DNA cut at the target locus, mediating integration of a therapeutic LSD donor cDNA. To ensure long-term expression of the transgenes in vivo we target the albumin locus as a “safe harbor” in hepatocytes via co-delivery of the albumin ZFNs and LSD donors by adeno-associated virus (AAV). This system exploits the high transcriptional activity of the native albumin enhancer/promoter; uses stably modified hepatocytes to potentially allow long-term expression of the inserted transgene; and utilizes an endogenous promoter obviating this requirement in the AAV payload. We have previously exploited AAV-mediated in vivo targeting of the murine albumin “safe harbor” locus for the synthesis of therapeutic levels of FVIII and FIX to overcome the clotting defect in hemophilic mice. Using the same approach, we co-delivered mouse albumin ZFNs with donor constructs encoding either human iduronate-2-sulfatase (IDS) deficient in Hunter syndrome, α-L-iduronidase (IDUA) for Hurler syndrome, or Glucocerebrosidase (GBA) for Gaucher disease via AAV in WT mice. We show stable integration of the LSD donors at the albumin locus, resulting in liver-specific expression and secretion of these proteins into plasma. This led to a 4-fold (GBA), 10-fold (IDUA) or 100-fold (IDS) increase in enzymatic activity in the plasma, demonstrating that the secreted proteins are functional. Importantly, increased activity was also detected in secondary tissues (spleen) showing efficient uptake and activity in distal tissues. Moreover, preliminary data in MPSI and MPSII mice suggests these levels of IDS and IDUA expression may lead to correction of the enzyme deficiency. IDS, IDUA and GBA expression remained stable throughout the study (up to 2 months), while expression of FIX has been stable for >1 yr suggesting this process results in long-term protein expression. In summary, our data provide proof of concept for ZFN-mediated targeting of the albumin locus in hepatocytes as an in vivo protein replacement platform to express different proteins associated with lysosomal storage diseases.

Expression, purification and characterization of zinc-finger nuclease to knockout the goat beta-lactoglobulin gene

Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products.

Genome Editing: Potential Treatment for Lysosomal Storage Diseases

The recent progress in genome editing technology using the engineered zinc finger nucleases (ZFN), transcriptional activator-like effector nucleases (TALEN), and more recently, clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) system have enabled the possibility of precisely modifying target sites in the genome. This technology brings hope of a cure for many genetic diseases. With this review, our goal is to discuss how targeted genome editing can be combined with hematopoietic stem cell transplantation and other approaches to be used for the treatment of a particular group of genetic diseases, the lysosomal storage disorders. We also highlight which diseases within this group would potentially benefit from this treatment and what are the main problems to be addressed to transform this promising technology into reality.

HPRT As a Selectable Safe Harbor for Transgenesis

A current limitation in gene therapy is obtaining a sufficient number of modified cells to produce a therapeutic effect in vivo. In several diseases, correction of a mutant allele confers a selective growth advantage to the modified cells, thus enhancing efficacy with moderate initial modification. For most diseases, however, there is no selective advantage to the corrected cells. One potential strategy to address this limitation is in vivo selection of modified cells using pharmacological agents. It has previously been shown that 6-thioguanine (6-TG), an FDA-approved chemotherapeutic small molecule, is cytotoxic to cells expressing the enzyme HPRT, allowing for selective growth of HPRT knockout cells. Knockout of HPRT can be achieved by creating a nonsense mutation in an upstream exon, or by terminating splicing by introducing a large transgene into an intron. To allow for selectable transgenesis of only cells which have undergone targeted integration (TI), engineered zinc-finger nucleases (ZFNs) were used to insert a virally-delivered transgene into an HPRT intron. After two weeks of in vitro 6-TG selection following genome modification, a 95-fold increase in TI was observed in pooled K562 cell populations to a final level of 72% TI, whereas a 30-fold increase in transgene-expressing live cells was seen in peripheral blood-mobilized primary CD34+ cells resulting in 90% transgene-positive live cells. Furthermore, a 72-fold increase in transgene mRNA transcript was observed after two weeks of erythroid differentiation and 6-TG selection of CD34+ cells compared to unselected genome-modified controls. These results represent an important step in developing hematopoietic stem cell (HSC)-based gene therapies, as well as a platform technology for creating gene-modified HSC populations with high proportions of therapeutic transgene expression via precise, targeted integration of a transgene of interest. DisclosuresConway:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Cost:Sangamo Biosciences: Employment.

Targeting CCR5 for anti-HIV research.

Highly active antiretroviral therapy (HAART) is the only approach for human immunodeficiency virus (HIV) infection treatment at present. Although HAART is effective in controlling the progression of infection, it is impossible to eradicate the virus from patients. The patients have to live with the virus. Alternative ways for the cure of HIV infection have been investigated. As the major co-receptor for HIV-1 infection, C-C motif chemokine receptor 5 (CCR5) is naturally an ideal target for anti-HIV research. The first CCR5 antagonist, maraviroc, has been approved for the treatment of HIV infection. Several other CCR5 antagonists are in clinical trials. CCR5 delta32 is a natural genotype, conferring resistance to CCR5 using HIV-1 strains. Gene therapy research targeting this mutant has been conducted for HIV infection treatment. A Berlin patient has been cured of HIV infection by the transplantation of stem cells from a CCR5 delta32 genotype donor. The infusion of an engineered zinc finger nuclease (ZFN)-modified autologous cluster of differentiation 4 (CD4) T cells has been proved to be a promising direction recently. In this study, the anti-HIV research targeting CCR5 is summarized, including CCR5 antagonist development, stem cell transplantation, and gene therapy.

Efficient generation of myostatin (MSTN) biallelic mutations in cattle using zinc finger nucleases.

Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle.

Generation of mastitis resistance in cows by targeting human lysozyme gene to β-casein locus using zinc-finger nucleases

Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine β-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in β-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP+/eGFP+) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

ZFN-Driven Gene Editing Prevents HLA-A Expression On Hematopoietic Stem Cells -Improving The Chance Of Finding An HLA-Matched Donor

Hematopoietic stem cells (HSCs) are administered (i) to restore hematopoiesis and immunity in the course of hematopoietic stem-cell transplantation (HSCT), (ii) as a replacement for inherited blood disorders and bone marrow failure, (iii) to regenerate cells of alternative lineages for restorative medicine, and (iv) as a source for generating specific hematopoietic cells (e.g., T cells, NK cells, and dendritic cells). However, the widespread application of allogeneic HSCs for humans is hampered by their immune-mediated destruction by host T cells recognizing mismatched HLA or by HLA-specific antibodies. Despite pre-banking umbilical cord blood (UCB) units and access to adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product is challenging for many recipients, especially those from ethnic minorities who are under-represented in the donor pool. The available donor pool would be markedly increased if donor HSCs were edited to eliminate expression of the HLA-A locus. Indeed, modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when matched for HLA-B, C and DR, instead of HLA-A, B, C and DR. We have previously shown that engineered zinc finger nucleases (ZFNs) can disrupt HLA-A expression in genetically edited T cells (Blood 2013). To extend this proof-of-concept to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus. CD34+lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB. Electro-transfer of in vitro transcribed mRNA encoding the HLA-A-specific ZFN generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1). As expected, SR1 treatment maintained greater numbers of CD34+ cells (also CD34posCD38neg) in culture compared to controls. DNA sequence analysis revealed that HLA-Aneg HSCs encode the expected nucleotide changes at the ZFN target site. An in vivo engraftment assay, using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, demonstrated that HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg hematopoietic cells (Figure). Thus, disruption of HLA-A expression in HSCs provides an appealing approach to increasing the chances for of finding HLA-matched donors and may broaden the clinical application of allogeneic HSCT. Furthermore, the ability to genetically edit HSCs has implications for (i) preventing immune-mediated recognition of HLA-disparate HSC and (ii) preventing immune mediated recognition of self-antigens.Engraftment of HLA-A2neg HSCs was evaluated in vivo. Data shown are flow-cytometry analysis of bone marrow obtained from NSG mice 16 weeks after HSC injection. HSC engraftment and HLA-A2 expression in NSG mice injected with un-modified HSCs (left panel) and HSCs treated with the HLA-A specific ZFNs (right panel) are shown. Data are gated on human CD45 positive cells. [Display omitted] Disclosures:Reik:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

Autologous Hematopoietic Stem/Progenitor Cell (HSPC) Therapy For Monogenic Blood Disorders: Scalable, cGMP-Compliant Process For Generating Highly Efficient Genome Edited HSPC

BackgroundAllogeneic bone marrow transplant (BMT) is the only curative method for a number of monogenic blood disorders, including various forms of hemoglobinopathies and severe combined immunodeficiencies. Aside from the significant hurdle of finding an identical HLA-matched related donor, allogeneic BMT recipients require chronic immunosuppression to mitigate the significant risk of GVHD and are at greater risk for graft failure. Autologous gene modified HSPC potentially provide a much safer alternative to allogeneic BMT and abrogate the need for finding HLA-matched donors. Here, we report the development of a highly efficient process for generating gene modified human HSPC at clinical-scale (>150 x 10^6 cells for 2x10^6/kg) using clinical-grade equipment. MethodsHealthy donors were administered Neupogen® (10mg/kg/day) for 4-5 consecutive days and then apheresed. Enrichment of CD34+ cells was performed with the Miltenyi CliniMACS® system. Genome editing was achieved via the introduction of mRNA encoding two engineered zinc finger nucleases (ZFN) using a scalable electroporation device. Cells were harvested and cryo-preserved with a controlled-rate freezer. Cell recovery and viability, gene modification efficiency, stem cell pluripotency and engraftment potential were evaluated by in vitro assays and in a humanized NSG mouse model. ResultsEnrichment of CD34+ cells from mobilized leukopak products was highly efficient with the Miltenyi CliniMACS® system (median recovery = 327 million CD34+ cells per 10L mobilized leukopak). The positively selected fractions were >98% CD34+ by FACS analysis. Two large scale electroporation devices (BTX AgilePulse Max® and MaxCyte GT®) were evaluated. Each device is capable of electroporating up to 300 million cells. The optimal transfection conditions for both devices were first identified by using a GFP mRNA to evaluate transfection efficiency by flow cytometry, which resulted in the identification of conditions (voltage and duration) that yielded gene transfer efficiencies of >90%. Compatibility of this protocol with driving endogenous gene modification was evaluated using mRNA encoding ZFNs that target various endogenous gene loci. Highly efficient levels of genome editing were observed in CD34+ HSPC each transfected with a different pair of ZFNs (median = 53%, 43%, 45% and 42% modified alleles at four distinct disease-relevant loci). At the optimal mRNA dose for each ZFN pair, cell viability post electroporation was >80%, comparable to untransfected controls. Process suitability was evaluated by in vitro colony forming cell assay. No significant differences in colony formation were observed between gene modified and untransfected control samples. The capacity of electroporated HSPC to engraft and support multi-lineage development of human hematopoietic cells was evaluated in NSG mice, and no differences were observed between the ZFN-treated and untransfected control cells. In addition, high levels of gene modification (19-28%) were detected in bulk human cells from the blood and tissues of engrafted mice, and in various sorted cell types (bone marrow CD34 and differentiated B and T cells). ConclusionWe have developed a scalable process capable of deriving >300 million gene-modified CD34+ HSPC. This process supports high levels of ZFN-driven genome editing, is well tolerated, and causes no discernable defect in the hematopoietic potential of these cells to develop into multiple cell lineages, with high gene editing levels maintained in the differentiated progeny of the HSPC. These results support the use of gene modified autologus HSPC for the treatment of monogenic blood disorders. Disclosures:Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Ya-Li:Sangamo BioSciences: Employment. Kim:Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Wang:Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Ando:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment.

ZFN Mediated Targeting Of Albumin “Safe Harbor” Results In Therapeutic Levels Of Human Factor VIII In a Mouse Model Of Hemophilia A

Genome editing utilizing engineered zinc finger nucleases (ZFNs) is a promising approach to achieve long-term expression of therapeutic genes in vivo. We have previously demonstrated in vivotargeting of the endogenous murine albumin locus as a “safe harbor” for high levels of protein production, resulting in sufficient Factor IX to correct the disease phenotype in hemophilia B mice. Targeted insertion of the donor sequence into the genome offers multiple advantages. First, we are able to exploit the high transcriptional activity of the native albumin enhancer/promoter. Second, by obviating the need for these regulatory elements within the donor, we expand the effective carrying capacity of adeno-associated viral (AAV) vectors to enable delivery of larger transgenes that may not package efficiently, such as coagulation factor 8.B-Domain Deleted Factor VIII (BDD-F8) cDNA is approximately 4.4kb. Inclusion of required enhancer/promoter elements results in a construct that exceeds the ideal packaging limitations of rAAV vectors. However, since these regions are not required for our gene editing approach the promoterless hBDD-F8 donor remained below the AAV packaging capacity. Importantly, intravenous delivery of 5e11 vg of AAV8-mAlb-ZFN and 5e11 vg of AAV8-BDD-F8-Donor to hemophilia A mice resulted in 54.6% (±4.1%) FVIII activity in the blood 2 weeks following administration - confirming the potential of the albumin locus to express high levels of the targeted transgene. To further optimize in vivo ZFN-based genome editing with a view toward the ultimate clinical use of this technology, we sought methods to further increase ZFN potency (and thus limit the dose of AAV necessary for function). Of several strategies pursued (e.g. codon optimization and inclusion of a intron in the expression cassette) the most successful was to deploy separate vectors expressing each individual ZFN rather than a single vector encoding a dual expression cassette carrying both ZFNs separated by a 2A fusion peptide. Using next generation sequencing (Illumina's MiSeq) technology to quantify insertions and deletions indicative of DNA cleavage and repair, we observed a >3-fold increase in ZFN potency in vivo by transitioning from the dual expression vector to two individual ZFN vectors at equivalent total vector doses. Given the encouraging results obtained in mice, we next sought to examine the effectiveness of targeting the albumin locus in non-human primates (NHPs). Importantly, a single intravenous co-injection of two individual AAV vectors encoding each of the NHP targeted albumin-specific ZFNs resulted in persistent levels of gene modification in liver biopsies from treated Rhesus macaques - demonstrating successful in vivocleavage in a large animal model.These data support the use of ZFN technology in the targeting of endogenous loci with large therapeutic transgenes that are not ideally suited for episomal AAV based expression (such as F.VIII). Together our results support the further investigation of genome editing at the albumin locus as a novel method for in vivo protein replacement. Disclosures:Doyon:Sangamo BioSciences, Inc.: Employment. Wechsler:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. High:Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Intrexon: Consultancy; Genzyme, Inc.: Membership on an entity's Board of Directors or advisory committees; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shire : Consultancy; Benitec: Consultancy.

Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases

Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probability of identifying ZFN-mediated positive targeted disruption events is small. In this paper, we used the context-dependent assembly method to synthesize two pairs of ZFNs targeted to the sheep MSTN gene. We verified the activity of these ZFNs using an mRFP-MBS-eGFP dual-fluorescence reporter system in HEK293T cells and, according to the expression level of eGFP, we obtained a pair of ZFNs that can recognize and cut the targeted MSTN site in the reporter vector. The activity of ZFN was increased by cold stimulation at 30 °C and by mutant the wildtype FokI in ZFN with its counterpart Sharkeys. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two MSTN mutant cell lines, identified by flow cytometry and sequencing, were obtained.

Zinc Finger Nuclease-Expressing Baculoviral Vectors Mediate Targeted Genome Integration of Reprogramming Factor Genes to Facilitate the Generation of Human Induced Pluripotent Stem Cells

Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinase-mediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.