Randall K. Merling

Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.

We report the design of a unique two‐plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV‐G pseudotyped vector derived from HIV‐1 that expresses antisense to the HIV envelope gene. We found that a two‐plasmid approach to production resulted in higher vector production titers when compared with a three‐plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication‐competent lentivirus (RCL). A native cis‐acting ribozyme is used to prevent read through into the envelope gene from the upstream gag‐pol genes in the packaging vector, thus preventing RNAs containing gag‐pol and env together for comparable safety to a three‐plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID‐hu mouse transplantation model, which correlates with the presence of an anti‐HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one‐step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation. Copyright

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.

An AAVS1-Targeted Minigene Platform for Correction of iPSCs From All Five Types of Chronic Granulomatous Disease

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Generation of Functionally Mature Neutrophils from Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are pluripotent stem cells established from somatic cells. The capability of iPSCs to differentiate into any mature cell lineage under the appropriate conditions allows for modeling of cell processes as well as disease states. Here, we describe an in vitro method for generating functional mature neutrophils from human iPSCs. We also describe assays for testing these differentiated cells for neutrophil characteristics and functions by morphology, cell surface markers, production of reactive oxygen species, microbial killing, and mobilization of neutrophils to an inflammatory site in an in vivo immunodeficient mouse infusion model.

Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four‐stage differentiation protocol involving: embryoid body (EB) formation (stage‐1); EB culture with hematopoietic cytokines (stage‐2); HSPC expansion (stage‐3); and neutrophil maturation (stage‐4). CD34+CD45− putative hemogenic endothelial cells were observed in stage‐3 cultures, and expressed VEGFR‐2/Flk‐1/KDR and VE‐cadherin endothelial markers, GATA‐2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC‐associated microRNAs miR‐24, miR‐125a‐3p, miR‐126/126*, and miR‐155. Upon further culture, CD34+CD45− cells generated CD34+CD45+ HSPCs that produced hematopoietic CFUs. Mid‐stage‐3 CD34+CD45+ HSPCs exhibited increased expression of GATA‐2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC‐associated microRNAs, and failed to engraft in immune‐deficient mice. Mid‐stage‐3 CD34−CD45+ cells maintained PU.1 expression and exhibited increased expression of hematopoiesis‐associated miR‐142‐3p/5p and a trend towards increased miR‐223 expression, indicating myeloid commitment. By late Stage‐4, increased CD15, CD16b, and C/EBPɛ expression were observed, with 25%‐65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. Stem Cells 2016;34:1513–1526

Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

Pseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in NCF1, a gene with 2 pseudogenes, NCF1B and NCF1C. The most common NCF1 mutation, a GT deletion (ΔGT) at the start of exon 2 (>90% of alleles), is constitutive to NCF1B and NCF1C. NCF1 ΔGT results in premature termination, undetectable protein expression, and defective production of antimicrobial superoxide in neutrophils. We examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases targeting the exon 2 ΔGT in induced pluripotent stem cells or CD34+ hematopoietic stem cells derived from p47-CGD patients. Correction of ΔGT in NCF1 pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.

557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30-kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPR-mediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB2-13 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.

527. Improvement of Pre-Clinical Non-Human Primate Model for Pluripotent Stem Cell Based Therapies by Introducing Marker Genes in Safe Harbor Locus

Induced pluripotent stem cells (iPSCs) are being developed as sources for clinical cellular regenerative therapies, as well as valuable in vitro human disease models. Derivation of iPSCs from non-human primates (NHP) affords the opportunity to test the safety, feasibility and efficacy of proposed iPSC-derived cellular delivery in species with physiology, immunology and scale similar to humans. However, there is a need for stable and safe labeling methods for iPSCs and their differentiated progeny allowing analysis of survival, proliferation, tissue integration and biodistribution, in vitro and in vivo. Typically, marker genes have been inserted into target cells by transduction with randomly-integrating viral vectors. However, these methods raise concerns regarding genotoxicity and transgene silencing, particularly in pluripotent stem cells, limiting their utility for tracking and eventual clinical applications. Targeted integration into genomic “safe harbors” offer a promising alternative approach to mark target cells, potentially circumventing these issues. The adeno-associated virus integration site 1 (AAVS1) has been proposed as a suitable safe harbor for human cells, and we now investigate its utility in our rhesus macaque NHP iPSC model. We have efficiently knocked-in both a truncated CD19 (hΔCD19) marker gene a non-immunogenic and clinical relevant marker, or green fluorescent protein (GFP) at the homologous AAVS1 site in rhesus iPSCs (RhiPSCs) using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) system. PCR and Southern blot analyses demonstrated highly efficient knock-in into the AAVS1 locus, with over one third of clones screened containing only targeted but not random integrations. (Table 1Table 1). Edited RhiPSC-GFP/hΔCD19 clones retained a normal karyotype and pluripotency - as shown by teratoma formation. Directed differentiation of these clones to neutrophils, hepatocytes or cardiomyocytes was not hindered by the knock-in of marker genes into the AAVS1 sites. Notably, transgene expression was stable in undifferentiated RhiPSCs and differentiated cell types derived from the RhiPSC (Figure 1Figure 1), in contrast to prior experience with viral vector delivery. We have established a computational platform to assess off-target effects of guide RNAs in the rhesus genome. Genetically marked RhiPSCs afford a unique opportunity to develop clinically relevant models for iPSC-based cell therapies.Table 1Summary of CRISPR-mediated gene editing in rhesus iPSCsOriginal iPSC cloneReporter geneClones with TI/Clones screened1Clones without RI/Clones with TI2ZG15-M11-10hΔCD194/42/8GFP14/145/9ZG32-3-4hΔCD19ND1/4GFPND2/4ZH26-HS41hΔ CD19ND1/4Total18/18(100%)11/29 (37.9%) View Table in HTML RI: random integration, TI; targeted integration, ND: not determined1based on by PCR analysis2based on Southern blot analysisView Large Image | Download PowerPoint Slide

57. Seamless Targeted Correction of CYBB Exon 5 Mutations Restores Granulocyte Function in X-Linked Chronic Granulomatous Disease iPSCs

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted “safe-harbor” gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with “excision-only” piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.

332. ZFN-Mediated Minigene or Dinucleotide Gene Correction of p47phox Deficient Autosomal Recessive Chronic Granulomatous Disease iPSC to Generate Oxidase Functional Neutrophils

Chronic granulomatous disease is an immune deficiency caused by defective phagocyte NADPH oxidase resulting in severe infections and autoimmune complications. NCF1 gene mutations resulting in p47phox deficiency are the most common cause of autosomal-recessive chronic granulomatous disease (AR-CGD), and notably, >80% of patients have homozygous GT-deletions in exon 2, resulting in a frame shift, premature termination, and absence of p47phox expression. Here we show two nucleofection strategies for NCF1 gene correction using zinc-finger nuclease (ZFN) targeting the NCF1 locus in patient EBV-immortalized B cells and in induced pluripotent stem cells (iPSC) derived from p47phox-deficient patients. With the first strategy, ZFN-mediated cut at start of exon 2 allows insertion of a minigene (NCF1 cDNA with deleted exon 1) to allow continuous transcription from the endogenous NCF1 exon 1 to the donor minigene exon 2 until the end of the cDNA and beta-globin polyadenylation signal. The second strategy takes advantage of the most prevalent GT-deletion by providing a dinucleotide corrective donor plasmid for gene editing using the same ZFNs. The two donors contain ~750 bp to 1000 bp flanking homology arms and also encode a selectable marker: a puromycin resistance gene flanked by loxP sites to allow for excision after selection. In patient EBV-immortalized B cells, minigene and dinucleotide targeted gene correction of constitutively active NCF1 was 8-20% after puromycin-selection from obtained clones. The rates of correction in iPSC were generally lower, less than 2%. Use of AAV2 vector to deliver dinucleotide corrective donor to iPSC increased insertion efficiency to >80% after puromycin selection. Gene corrected B cells for both correction strategies restored ROS function, shown by chemiluminescence assay. Neutrophils differentiated from gene corrected iPSCs demonstrated correction of oxidase activity by chemiluminescence or dihydrorhodamine flow cytometry assay. With both methods, p47phox protein expression is seen in neutrophils differentiated from iPSC but not in undifferentiated iPSC, demonstrating cell type-specific regulation of expression at the corrected locus. In summary, we show that iPSC generated from CGD patient cells can be gene corrected through a targeted dinucleotide correction method for greater than 80% of alleles and minigene correction for mutations other than the GT-deletions.