690. Permanent Epigenetic Silencing of Human Genes With Artificial Transcriptional Repressors

There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship

6. Targeted Genome Editing of Cell Lines for Improved and Scalable Production of Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. The manufacturing of clinical-grade LVs relies on transient transfection of vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic and antigenic class-I major histocompatibility complex (MHC-I) expressed on LV packaging cells and subsequently incorporated on the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of the MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. We generated B2M-negative cells devoid of surface-exposed MHC-I, which retain the ability to produce LVs. In order to insert the LV genome of interest in the packaging cell line, we performed site-specific integration in predetermined loci of the genome of these cells, chosen for robust expression, exploiting artificial nucleases and homology-directed repair. In several independent iterations of this process, we generated producer cell lines both for LV expressing marker genes and a therapeutic gene, i.e. coagulation factor IX (FIX), the gene mutated in hemophilia B. We show that these LV producer cells are stable in culture and can produce several liters of LV-containing conditioned medium. These LVs have comparable or only slightly lower infectious titer and specific infectivity than LVs produced by state-of-the-art transient transfection process and can transduce therapeutically relevant target cells, such as hematopoietic stem/progenitor cells and T lymphocytes to high efficiency. Moreover, we intravenously administered FIX-expressing LVs produced by the cell line to hemophilia B mice and established therapeutic levels of circulating FIX. These data indicate that site-specific integration is an efficient, rapid and reproducible method to generate LV producer cells, starting from a universal stable inducible LV packaging cell line. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the quality, safety and sustainability of LV production for clinical use

286 genome editing of inducible cell lines for scalable production of improved lentiviral vectors for human gene therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. Current manufacturing of clinical-grade LVs mostly relies on transient transfection of plasmids expressing the multiple vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. These LV packaging cells are stable in culture even after single-cell cloning and can be scaled up to large volumes. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic class-I major histocompatibility complexes (MHC-I) expressed on LV packaging cells and incorporated in the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. Strikingly, the MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with syngeneic monocytes exposed to LV, from several (n=7) healthy donors. To reproducibly generate LV-producer cell lines from these cells, we insert the LV genome of interest in the AAVS1 locus, chosen for robust expression, exploiting engineered nucleases and homology-directed repair. By this strategy, we have obtained several independent producer cell lines for LVs that express marker or therapeutic genes and are devoid of plasmid DNA contamination. LVs produced by these cells reproducibly show titer and infectivity within the lower bound range of standard optimized transient transfection, and effectively transduce relevant target cells, such as hematopoietic stem/progenitor cells and T cells ex vivo and the mouse liver in vivo. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the yield, quality, safety and sustainability of LV production for clinical use

130 purification of large scale mrna encoding zfn nucleases by dhplc technology

A novel strategy of targeted gene correction of the interleukin-2 receptor common gamma chain (IL2RG) gene for the treatment of X-linked Severe Combined Immunodeficiency (SCID-X1) is achieved by the combination of a pair of IL2RG-specific Zinc Finger Nucleases (ZFN) and the correct-gene template DNA delivered by integration-defective lentiviral vector (IDLV).The transient expression of the ZFN pair targeting the disease-causing gene is obtained by the electroporation of the two corresponding mRNAs, produced by in vitro transcription starting from plasmid DNA template. A major limitation of the mRNA transcribed in vitro is the presence of residual contaminants such as short RNAs and double stranded (ds)RNAs that may affect the function and spectrophotometric quantification of the product hampering therefore the delivery of high quality and precise amount of mRNA to target cells. Moreover, dsRNA contaminants represent a possible risk in terms of immunogenicity of the product, leading to activation of unwanted innate immune response with consequent reduction/abrogation of mRNA translation as well as potential alteration of the properties of the transfected cells. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we combined different strategies: (i) inclusion of UTRs and polyA tails in the DNA template used for mRNA production; (ii) use of modified nucleotides during mRNA production and (iii) purification of the mRNAs by dHPLC with a reverse phase column made of non-porous matrix consisting of polystyrene-divinylbenzene copolymer beads alkylated with C-18 chains (Transgenomic, LTD.). In particular, the purification of in vitro transcribed mRNAs by means of dHPLC has been shown to strongly improve the translation of mRNA and significantly reduce the contaminant presence thus preventing innate immunity and eventually increasing modified cells persistence in vivo. We have developed feasible and reproducible, small and large scale mRNA production and downstream purification processes of the ZFN pairs obtaining accurate RNA quantification and reduced risk of immunogenicity. The full process achieved a 60% yield, loading with a 500µg RNA for each run with a single clean chromatographic peak. Furthermore, the level of residual organic solvent (i.e. Acetonitrile) used in the purification process is compatible with that applicable into clinic. The highly translatable non-immunogenic dHPLC-purified mRNA can be delivered without toxicity and represents a powerful and safe tool for the application of gene therapy protocols

現地観測に基づく河川流の乱流特性に関する研究

博士（工学）神戸大

Dynamic Activity of miR-125b and miR-93 during Murine Neural Stem Cell Differentiation In Vitro and in the Subventricular Zone Neurogenic Niche.

Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function

Activity of miR-124 and miR-23a in NSC-derived neurons and astrocytes.

<p>Qualitative and quantitative GFP expression in neurons (βtubIII, Map2; blue) and astroglial cells (GFAP, nestin; blue) in bdLV.CTRL-, bdLV.miRT124- and bdLV.miRT23a-transduced NSC-derived <i>differentiated cells</i> (10 days in vitro). (<b>A</b>) Transduced cells (red, anti-mCherry antibody) in bdLV.CTRL-transduced cultures express bright GFP (green; direct fluorescence). (B–E) A significant decrease of GFP expression is observed in bdLV.miRT124-transduced neurons (βtubIII, MAP2) (<b>B, C</b>) and in bdLV.miRT23a-transduced astrocytes (GFAP) and immature glial cells (nestin) (<b>D, E</b>). Arrowheads indicate GFP⁺marker⁺ (miR^−/low) cells, arrows indicate GFP⁻marker⁺(miR^+/high) cells. Data are the mean ± SEM; n = 3 experiments, 1–3 coverlips/antigen/experiment. Data for each marker in bdLV.miRT-transduced cells were compared to their counterpart in bdLV.CTRL-transduced cells using One-way analysis of variance followed by Bonferroni’s posttest. * p<0.05, *** p<0.001. Scale bars, 50 µm.</p

Activity of miR-124, miR-23a, miR-125b and miR-93a in striatal cell types.

<p>(<b>A</b>) Cell type composition (NeuN, neurons; GFAP, astrocytes) quantified by confocal IF analysis in striatal tissues of PND40 untransduced (UT) mice<b>.</b> (<b>B</b>) Quantitative analysis and representative confocal images of GFP expression (green, direct fluorescence) and immunoreactivity for NeuN (neurons, red) and GFAP (astrocytes, red) in brain tissue sections of PND40 mice after neonatal striatal injection of LV.CTRL (<b>C</b>), LV.miRT124 (<b>D</b>), LV.miRT23a (<b>E</b>)<b>.</b> Nuclei are counterstained with TO-PRO-3 (blue). CC, corpus callosum; CPu, Caudate Putamen<b>;</b> ctx, cortex. Scale bars: 100 µm (C–E). Data are the mean ± SEM; n = 3 animals per experimental group, 2–4 sections/animal. Data were analyzed by one-way analysis of variance followed by Bonferroni’s posttest. *** p<0.001 (NeuN), ∧∧ p<0.01 (GFAP) versus NeuN and GFAP values of LV.CTRL-injected mice. (<b>F, G</b>) Quantification and representative images of miR-125b activity after striatal injection of bdLV.miRT125b. Grey bars indicate the percentages of GFP⁺mCherry⁺ cells in bdLV.CTRL-injected mice. (<b>H, I</b>) Quantification and representative images of miR-93 activity after striatal injection of bdLV.miRT93. Arrowheads indicate GFP⁺ (miR^−/low) cells; arrows indicate GFP⁻ (miR^+/high) cells. Data are the mean ± SEM; n = 3 animals per experimental group, 2–4 sections/animal. Data were analyzed by one-way analysis of variance followed by Bonferroni’s posttest. **p<0.01, ***p<0.001 versus bdLV.CTRL. Scale bars: 50 µm (G, I).</p

Modulation of miR-125b and miR-93 activity in the SVZ neurogenic niche.

<p>(<b>A</b>) Schematic of the neurogenic pathway in adult mice. From the SVZ stem cell niche, newly generated neuroblasts migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB), where they integrate as mature neurons. Red lines indicate the levels of the sections analyzed in this study<b>.</b> DG, dentate gyrus of the hippocampus. (<b>B</b>) Representative confocal images of PND40 mice at the level of the SVZ, RMS, OB after injection of LV.CTRL at PND2. Note the distribution of GFP⁺ neurons in the different OB layers (form dorsal to ventral: GlL, glomerular layer; ExPl, external plexiform layer; IPL, internal plexiform layer; MiL, mitral cell layer; GCL, granule cell layer; Me, medulla). CC, corpus callosum; CPu, Caudate Putamen; lv, lateral ventricle. Scale bars, 300 µm. (<b>C</b>) LV-marked cell type composition quantified at PND40 in the SVZ of LV.CTRL and bdLV.miRT-injected mice. (<b>D</b>) Downregulation of GFP expression (direct fluorescence) in the transduced (mCherry⁺) nestin⁺, GFAP⁺ or βTubIII⁺ cells in bdLV.miRT-injected mice when compared to LV.CTRL-injected mice. (<b>E</b>) Representative confocal pictures of the SVZ of PND40 mice showing robust downregulation of GFP expression in transduced (mCherry⁺) nestin⁺ and GFAP⁺ cells in LV.miRT125b and LV.miRT93-injected mice when compared to LV.CTRL-injected mice. Arrowheads indicate GFP⁺mCherry⁺marker⁺ (miR^−/low) cells; arrows indicate GFP⁻ mCherry⁺marker⁺(miR^+/high) cells. Scale bars, 300 µm (<b>F</b>) Distribution of transgene-labelled neurons in the different OB layers (legend as in panel B). (<b>G</b>) Downregulation of GFP expression in the transgene-labelled neuronal population of bdLV.miRT-injected mice indicates modulation of miR-125b and miR-93 activity in different OB layers. Data are the mean ± SEM. We analyzed 2–3 OB sections/mice, n = 2–4 mice/treatment group (300–3000 transduced cells). Each bdLV.miRT-treated group was compared to the LV.CTRL group by One-Way analysis of variance followed by Dunnet’s Multiple comparison test, *p<0.05, ** p<0.01, *** p<0.001 vs LV.CTRL. (<b>H</b>) Representative confocal pictures showing downregulation of GFP expression (direct fluorescence) in the transduced (mCherry⁺) neurons in the superficial (upper panel) and deeper (lower panel) OB layer in bdLV.miRT-injected mice when compared to LV.CTRL-injected mice. Scale bars, 150 µm.</p

Lineage-specific modulation of miR-125b and miR-93 activity during NSC differentiation.

<p>(<b>A</b>) Representative confocal images of bdLV.CTRL-transduced NSC cultures (mCherry⁺; red, anti-mCherry antibody) showing bright GFP expression (green; direct fluorescence) in neurons (βtubIII, blue) and astroglial cells (nestin, blue). (<b>B,C</b>) Quantification and representative images of miR-125b activity in glial and neuronal subpopulations in bdLV.miRT125b-transduced NSC cultures. (<b>D,E</b>) Quantification and representative images of miR-93 activity in the glial and neuronal subpopulations in bdLV.miRT93-transduced NSC cultures. Data are the mean ±SEM; n = 2 experiments, 2–5 coverlips/antigen/experiment. Prec, <i>stem/precursors</i>; Prog, <i>progenitors</i>; Diff, <i>differentiated cells</i>; d, days in vitro. Arrowheads indicate GFP⁺marker⁺ (miR^−/low) cells; arrows indicate GFP⁻marker⁺ (miR^+/high) cells. Scale bars: 50 µm. Data were analyzed by one-way analysis of variance followed by Bonferroni’s posttest. *, ∧, § p<0.01 versus bdLV.CTRL-transduced GFAP⁺ cells, nestin⁺ and βtubIII⁺ cells, respectively.</p