Olivier Humbert

The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model.

Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well.

In Vivo Hematopoietic Stem Cell Transduction

Current protocols for hematopoietic stem cell (HSC) gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. In vivo HSC gene therapy can be achieved by the direct modification of HSCs in the bone marrow after intraosseous injection of gene delivery vectors. A recently developed approach involves the mobilization of HSCs from the bone marrow into peripheral the blood circulation, intravenous vector injection, and re-engraftment of genetically modified HSCs in the bone marrow. We provide examples for in vivo HSC gene therapy and discuss advantages and disadvantages.

A Nonhuman Primate Transplantation Model to Evaluate Hematopoietic Stem Cell Gene Editing Strategies for β-Hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo gene editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

Rapid immune reconstitution of SCID-X1 canines after G-CSF/AMD3100 mobilization and in vivo gene therapy.

Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.

Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2

CD33 has long been pursued as immunotherapeutic target in acute myeloid leukemia (AML) [1, 2]. Improved survival with gemtuzumab ozogamicin (GO) validates this approach [3]. Partly stimulated by GO’s success, several investigational CD33-directed therapeutics are currently in clinical testing [4]. However, CD33 expression on normal hematopoietic cells leads to “on-target, off-leukemia” toxicity with significant morbidity/mortality from profound cytopenias, limiting the use of CD33-directed immunotherapies [4]. This toxicity should be minimal if normal blood cells did not express the epitope targeted by these antibodies. Supporting the feasibility of CD33-engineering the hematopoietic system are the findings that CD33-deficient mice have a very mild phenotype and show no difference in cellular response to pro-inflammatory stimuli compared to wild-type animals, indicating functional degeneracy between CD33 and other proteins [5]. Moreover, recent studies have shown that CRISPR/Cas9-mediated disruption of the CD33 coding region in CD34+ hematopoietic stem and progenitor cells (HSPCs) may not affect engraftment [6], suggesting that the generation of CD33-manipulated hematopoiesis is a clinically viable strategy to protect from “off-leukemia” cell toxicity of CD33-directed immunotherapy. Here we have investigated an alternative, precise CD33 genome-editing approach that would only eliminate exon 2 and therefore the V-set immunoglobulin-like domain, which is the target of all current clinical CD33directed approaches. Our editing strategy is expected to result in expression of a naturally occurring shorter isoform of CD33 (CD33) but not full-length CD33 (CD33), which may minimize potential adverse effects associated with disruption of the entire CD33 locus. We used CRISPR/ Cas9 [7–10] to accomplish this goal and functionally assessed genome-edited human hematopoietic cells in vitro and in immunodeficient mice. Human myeloid ML-1 cells and human fetal liver CD34 + HSPCs were used for our studies. ML-1 cells were maintained as described [11]. Human fetal liver CD34+ cells were enriched by immunomagnetic separation from tissue obtained from Advance Bioscience Resources Inc. (ABR, Alameda, CA). Cells were cultured in StemSpan SFEMII media (StemCell Technologies, Cambridge, WA) supplemented with penicillin/streptomycin (Life Technologies, Carlsbad, CA), Stem cell factor , Thrombopoietin (both PeproTech, Rocky Hill, NJ), and FLT3-L (Miltenyi Biotec, Auburn, CA). CRISPR/Cas9-editing was carried out by electroporation of purified Cas9 protein (TrueCut Cas9 V2; ThermoFisher Scientific, Waltham, MA) complexed with synthetic guide RNAs (sgRNAs; Supplementary Table 1), which were modified at the 5′ and 3′ ends with 2′O-methyl-3′-phosphorothiate (Synthego, Redwood City, CA) using the ECM 380 Square Wave Electroporation system (Harvard Apparatus, Cambridge, MA) [12]. For evaluation of colony-forming units (CFUs), 1500 CD34+ cells were seeded in 3.5 mL ColonyGEL 1402 (ReachBio, Seattle, WA) and scored after 12–14 days. CFU DNA was extracted in QuickExtract (Epicentre, Madison, WI). We quantified drug-induced cytotoxicity as described previously [11, 13]. Briefly, parental and CRISPRengineered ML-1 cells were incubated in 96-well round These authors contributed equally: Olivier Humbert, George S. Laszlo and Hans-Peter Kiem, Roland B. Walter

284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain

X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the common gamma chain, γC (interleukin-2 receptor subunit gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.

288. Development of Cocal Glycoprotein Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T Cells

Lentiviral vectors (LVs) are routinely used for stable gene transfer and have demonstrated great promise in hematopoietic stem cell gene therapy and also immunotherapy using genetically modified T cells. LVs are commonly pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), which confers broad tropism to the vector and allows for vector concentration by centrifugation. However, the use of VSV-G has several limitations, such as susceptibility to inactivation by human serum complement making it unsuitable for in vivo delivery. Furthermore, VSV-G is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. In this study, we first validate the use of cocal vesiculovirus envelope to pseudotype LVs by demonstrating that cocal LVs transduce hematopoietic stem cells and CD4+ T cells more efficiently than VSV-G LVs. We also provide evidence that cocal and VSV-G envelopes use the same receptor for cell entry. We then describe the development of two high-titer, cocal-pseudotyped, LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T cell receptor (TCR). The different 3rd generation lentiviral helper genes were sequentially introduced in HEK293T cells by co-transfection with plasmids encoding antibiotic resistance genes followed by selection to allow for stable protein expression. Cells expressing the cocal envelope produced over 10-times more infectious LV particles as compared to VSV-G expressing cells. High-titer cocal producer cells were isolated by screening for best single clones, which were capable of generating concentrated titers above 108 infectious units per mL. We found that these producer cells were stable after serial passages for over 3 months, with no drop in titer detected over time. The resulting GFP and WT1-TCR vectors performed at least as well as identical vectors made with our standard transient transfection protocol for the transduction of CD34+ and CD4+ T cells, respectively. Cocal LV producer cells were also adapted for growth in suspension, serum-free culture, which will facilitate efforts for the scaling up of vector production. In summary, we have successfully developed two independent LV producer cells lines with clinically usable titers. The broad applicability of our cocal packaging cell line offers a promising tool toward the generation of large-scale, clinical grade LV.

758. A Nonhuman Primate Transplantation Model to Evaluate Gene Editing Strategies Aimed at Inducing Fetal Hemoglobin Production for the Treatment of Hemoglobinopathies

Fetal hemoglobin (HbF) is the major form of hemoglobin present in newborns but is almost completely replaced by adult hemoglobin after birth, where it constitutes less than 1 percent of total hemoglobin. Hereditary persistence of HbF is linked to mutations at multiple genetic loci that regulate the switch from fetal to adult hemoglobin. The targeting of these genes using site-specific nucleases thus constitutes a promising approach for the treatment of hemoglobinopathies by increasing HbF production.We have developed a nonhuman primate (NHP) model to investigate gene editing strategies aimed at inducing HbF production following hematopoietic stem cell (HSC) transplantation. As proof of principle, we focused on the transcription factor B-cell lymphoma/leukemia 11A (BCL11A), which functions as suppressor of HbF in humans. We disrupted the Bcl11a coding region using Transcription Activator-Like Effector Nucleases (TALENs) and achieved on average 30% gene editing by electroporation of mRNA in NHP CD34+ cells. Erythroid differentiation of these cells in culture confirmed that HbF expression was increased in Bcllla-edited cells as compared to control cells. To determine if Bcl11a-edited HSCs could engraft and give rise to HbF-producing erythrocytes, we transplanted a NHP with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. Using next generation sequencing, we detected about 1 % disruption in vivo one week after transplant, to reach a set point of about 0.3% over the course of the experiment. We were able to track several clones that persisted at least 200 days post transplantation based on their mutation signatures, suggesting engraftment of Bcllla-modified cells. HbF production was monitored in this animal by flow cytometry analysis of peripheral blood and was compared with three transplanted controls and one untransplanted control. In all transplanted animals, we observed a rapid increase in the frequency of F cells, reaching 10% to 40%, and lasting for about 140 days. In contrast, F cell production in the untransplanted control remained constant and minimal (<0.5%). After returning to basal levels, we found significantly higher HbF levels (1-1.5%) in the animal transplanted with Bcl11a-edited cells as compared to all other transplanted animals. These findings were confirmed by real-time PCR analysis of hemoglobin transcripts, which showed a 5-to 10-fold increase in gamma to beta globin ratio in the animal transplanted with Bcl11a-edited cells as compared to all controls. We also initiated work demonstrating the targeted integration of the chemoselection cassette P140K/MGMT at the Bcl11a locus in NHP HSCs by co-delivery of TALEN mRNA with a donor template carried on an adeno-associated viral vector, offering the potential for in vivo selection of modified cells. In summary, our experiments establish the NHP as pre-clinical model to evaluate therapeutic gene editing strategies for the treatment of hemoglobinopathies.