Knock-in gene correction of induced pluripotent stem cells from pyruvate kinase deficient patient

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-11-2013La Deficiencia en Piruvato Quinasa (DPQ) es una enfermedad rara causada por mutaciones en el gen PKLR que provoca Anemia Hemolítica no Esferocítica Crónica (AHNEC). El único tratamiento definitivo para los casos graves de DPQ es el Trasplante Alogénico de Médula Ósea (TAMO). Debido a los riesgos asociados a TAMO, como la enfermedad de injerto contra huésped, y la baja disponibilidad de donantes adecuados, hacen que la terapia celular autóloga sea una alternativa atractiva para el tratamiento de los casos graves de DPQ. Una alternativa prometedora para el tratamiento de trastornos hereditarios hematopoyéticos es la combinación de la generación de células madre pluripotentes inducidas (iPSCs) a partir de muestras de pacientes, junto con la corrección especifica de sitio mediada por Recombinación Homóloga (RH). Con el fin de evaluar esta posibilidad como alternativa terapéutica para DPQ, hemos generado iPSC derivadas de Células Mononucleares de sangre periférica (CMN) de pacientes con DPQ (DPQiPSCs) mediante vectores virales basados en el virus Sendai y las hemos corregido mediante RH específica de sitio asistida por dos tipos de nucleasas de ADN, Meganucleasas (MG) y TALE nucleasas (TALENTM). Para ello, hemos seguido una estrategia de Knock-in en el locus PKLR. Se obtuvieron diferentes clones de hiPSC a partir de dos pacientes de DPQ y un donante sano, de los cuales se confirmó la pluripotencia y la desaparición de los vectores de reprogramación. Con el fin de evitar la reprogramación de células linfoides, se llevó a cabo una estrategia basada en el uso de citoquinas, verificándose mediante el análisis de reordenamientos somáticos genómicos característicos de linfocitos B y T. Es importante señalar que la diferenciación eritroide de DPQiPSCs es deficiente tal como ocurre en los pacientes con DPQ, manifestando una acumulación de células eritroides inmaduras positivas para los marcadores CD71 y CD235a, lo que confirma el potencial uso de hiPSC para el modelado de enfermedades. Con el fin de restaurar el defecto genético, se han utilizado MG y TALENTM para facilitar la introducción de parte del ADNc de RPK terapéutico en el intrón 2 del gen PKLR. La MG presentó una muy baja especificidad para el gen PKLR, sin embargo, después del uso de PKLR1 TALENTM, se consiguió una correcta integración en un alto porcentaje de clones analizados mediante PCR y Southern Blot, así como la presencia de la proteína terapéutica RPK recombinante. Sorprendentemente, debido a la presencia de un polimorfismo en un único nucleótido en el ADN genómico de uno de los pacientes, se detectó exclusivamente integración específica en uno de alelos. Se examinó la integridad del genoma mediante el análisis de mutaciones somáticas y de Variaciones de Número de Copia (VNC), detectándose tres mutaciones y seis VNC en la muestra de DPQiPSC corregida, que no estaban presentes en la muestra de CMN de sangre periférica original. Estas modificaciones no afectan a ningún gen que confiera ninguna ventaja selectiva durante el cultivo de hiPSC ni al uso de nucleasas. Por último, cabe destacar que las células DPQiPSC corregidas, una vez diferenciadas a al linaje eritroide, muestran un perfil de maduración normal, similar al observado en las hiPSCs de CMN de un donante sano. Como conclusión, los resultados presentados muestran la posibilidad de generar eritrocitos genéticamente corregidos específicamente en el locus PKLR, a partir de hiPSC de pacientes de DPQ.Pyruvate Kinase Deficiency (PKD) is a rare disease caused by mutations in the PKLR gene that leads to Chronic Non-Spherocytic Hemolytic Anemia (CNSHA). The only definitive treatment for severe cases of PKD is allogeneic Bone Marrow Transplantation (BMT). The risks associated to BMT, such as graft versus host disease, together with the low availability of suitable donors, make autologous cell therapy desirable for this disease. Patient specific induced Pluripotent Stem Cells (hiPSC) coupled with targeted gene correction via Homologous Recombination (HR), is a promising alternative for the treatment of hematopoietic inherited disorders. In order to prove the feasibility of this therapeutic alternative for PKD, we have generated integration free iPSCs from Peripheral Blood Mononuclear Cells (PB-MNC) of PKD patients (PKDiPSCs) using Sendai based viral vectors, and have corrected them through a Knock-In approach in the PKLR locus by using two types of DNA nucleases, Meganucleases (MG) and TALE nucleases (TALENTM). Different hiPSC clones were obtained from two patients and one healthy donor; these hiPSC clones showed pluripotent characteristics even after the disappearance of reprogramming vectors. A strategy to avoid lymphoid cells reprogramming within PB-MNC was successfully applied as neither T nor B cell receptor rearrangements were found in any of the analyzed hiPSC lines. Interestingly, erythroid differentiation of PKDiPSC was impaired as occurs in PKD patients, showing an accumulation of immature CD71/CD235a double positive erythroid cells and assessing the use of hiPSC for disease modeling. To restore the genetic defect, specific MG and TALENTM were used to facilitate the Knock-In of a codon optimized RPK cDNA in the second intron of the PKLR gene. Whereas the MG generated DSB with very low specificity, after using the PKLR1 TALENTM, correct integration in PKLR locus was confirmed by PCR and southern blot, and the presence of the recombinant therapeutic RPK was assessed at the protein level. Surprisingly, allele specific integration due to the presence of a single nucleotide polymorphism was identified in one of the patients, pointing out its potential use in specific allele substitution. Genome integrity was examined by analyzing the appearance of de novo somatic mutations and Copy Number Variations (CNVs), detecting three single nucleotide variants and six CNVs in the corrected PKD2iPSC. The majority of them were already present before correction but not in the PB-MNC. These modifications did not include genes that were clearly associated either to a selective advantage or to the use of the nucleases. More importantly, gene corrected coPKDiPSCs displayed a normal erythroid maturation profile, similar to the one observed in wild-type hiPSCs. Overall, we show the feasibility of PKLR locus specific gene correction in patient specific iPSCs

Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics

Targeted gene therapy and cell reprogramming in Fanconi anemia

Altres ajuts: European Regional Development FEDER Funds, Italian Ministry of Health, Fondo de Investigaciones Sanitarias, Dirección General de Investigación de la Comunidad de Madrid S2010/BMD-2420, La Fundació Privada La Marató de TV3 121430/31/32, Marató de TV3 464/C/2012Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies

All replication-defective E1A12S mutants sensitise prostate cancer cells to mitoxantrone and docetaxel except the AdE1A1104 virus.

<p>A) Drug dose responses in each cell line were evaluated after infection with AdE1A12S, AdE1A1102, AdE1A1104, and AdE1A1108 mutants with AdGFP as negative control to determine changes in drug EC₅₀ values. All cell lines were infected at doses killing <10% of cells alone; PC-3 cells at 100 ppc (left panel), DU145 cells at 10 ppc (mid panel) and 22Rv1 cells at 2.5 ppc (right panel). Data represent averages ±SD, n = 4–5 independent experiments analysed by t-test comparing EC₅₀ values for each combination to that of drug alone, expressed as percentages, *p<0.05 and °p<0.01. B) EC₅₀ values for mitoxantrone were determined with and without simultaneous infection with viral mutants at 2.5, 10 and 100 ppc for 22Rv1, DU145 and PC-3 respectively, and with (grey bar) and without (black bar) the addition of the pan-caspase inhibitor zVAD-fmk at 25 µM. EC₅₀ values are expressed as percentages of mitoxantrone alone (Ctrl), averages ± SD, n = 3. C) Flow cytometry of cells infected with the AdE1A12S mutants or treated with mitoxantrone (50 nM) alone and in combination and analysed for tetramethylrhodamine uptake (TMRE) as an indicator of mitochondrial depolarisation and apoptosis induction. AdE1A12S, AdE1A1102, AdE1A1104, AdE1A1108, and AdGFP alone (solid arrow; all cell lines) and AdE1A12S, AdE1A1102, AdE1A1104 and AdE1A1108 in combination with mitoxantrone (dashed arrow; 22Rv1 cells). Data expressed as % apoptotic cells; percentages of cells that showed mitochondrial depolarisation, averages ± SD, n = 3. D) Cells infected with each mutant at 100 (PC-3) or 2.5 ppc (22Rv), mock infected and treated with or without mitoxantrone at 50 nM. Changes in cell cycle were analysed by flow cytometry at 24, 48 and 72 h after infection and drug treatment in PC-3 cells or after 48 h for 22Rv1 cells, one representative study (n = 3), *p<0.05 comparing G2/M-phase in combination treated vs mitoxantrone alone, °p<0.05 G2/M-phase for mitoxantrone vs mock treated.</p

EC₅₀ values for mitoxantrone and docetaxel in prostate cancer cells transfected with E1A12S or GFP expressing plasmids.

<p>Data from one representative experiment treated with mitoxantrone for 3 days after transfection with pcDNA plasmids expressing the respective proteins, n = 3.</p

The dl1102 mutant prolongs time to progression in combination with docetaxel in PC-3 xenografts <i>in vivo.</i>

<p>A) Animals with PC-3 subcutaneous tumor xenografts were treated with the <i>dl</i>1102 (filled triangle) or <i>dl</i>1104 (filled circle) mutants or mock treated with <i>dl</i>312 (filled square) at 1×10⁹ vp (i.t. injections on day 1, 3, and 5) with and without docetaxel at 10 mg/kg (D10; i.p. administration on day 2 and 8, open squares), and tumor growth was monitored. *p<0.05, treatments compared with mock and single-agent treatments (one-way ANOVA), p<0.05 for <i>dl</i>1102 alone compared to mock, n = 6. B). In a second study animals with PC-3 subcutaneous tumor xenografts were treated as above with the indicated suboptimal doses of mutants at 1×10⁹ vp and docetaxel at 10 mg/kg (D10) or the respective combinations. Median time to tumor progression (tumor volume >500 µl) was determined by Kaplan-Meier survival analysis (8–10 animals per group). *p<0.05, combination-treated compared with docetaxel. C) PC-3 cells infected with the indicated mutants and treated with docetaxel at four constant ratios; 0.5, 2.5, 12.5 and 62.5 ppc/nM drug (indicated by the wedges). CI values were calculated from isobolograms and CI≤0.9 were considered synergistic, averages ±SEM, n = 3, *p<0.05 vs the theoretical additive values (0.9</p

Synergistic cell killing with a replication-defective virus expressing the small AdE1A12S protein, in combination with cytotoxic drugs.

<p>A) Isobolograms generated from EC₅₀ values for combinations of the AdE1A12S mutant with mitoxantrone (Mit) or docetaxel (Doc) at four constant ratios (0.5. 2.5, 12.5 and 62.5 ppc/nM drug) in PC-3 and DU145 cells. The straight lines represent the theoretical values for additive effects and points below the line synergistic cell killing, one representative study (n = 3–4). B) Characterization of replication of the AdE1A12S, AdE1A1102, AdE1A1108 and AdE1A1104 mutants in PC-3, DU145 and 22Rv1 cells. Levels of viral replication determined by the limiting dilution assay (TCID₅₀) for replicating and replication-defective mutants with identical E1A-deletions except for the additional deletion of the CR3-domain in E1A12S. Cells were infected with each mutant at 100 ppc and harvested 72 h later, averages ±SD, n≥3. The non-replicating AdGFP mutant was used as a control in all assays, *p<0.001 for the replicating compared to the corresponding replication-defective mutant (t-test). C) qPCR analysis of cells infected as described for the replication assays and harvested 24, 48 and 72 h later. Total copy number at each time point was normalised to the copy numbers detected 3 h after infection in 10 ng of total DNA, averages ± SEM, n = 2–3. D) Viral replication in normal human primary bronchial epithelial cells (NHBE) determined by TCID₅₀ for Ad5wt, <i>dl</i>1102 and <i>dl</i>1104 mutants infected at 100 ppc, n = 3, *p<0.005.</p

Potent cell killing of prostate cancer cell lines by replicating E1A-deletion mutants in combination with mitoxantrone.

<p>A) The replicating viruses used in the study had intact E1A-region (E1A13S) except for the indicated deletions. The replication-defective mutants were based on the E1A12S construct with the same deletions as in the replicating viruses; AdE1A1102 (Δ26–35), AdE1A1104 (Δ48–60), AdE1A1108 (Δ124–127), in addition to deletion of the CR3-region, responsible for viral transcriptional activity. B) EC₅₀ values for the replicating mutants were determined from dose-response curves and presented as averages ± SD, n = 3. Significantly different values compared to Ad5 are indicated. C) Sensitization of the human PC-3, 22Rv1 and DU145 cells to mitoxantrone by fixed doses of each virus at EC₁₀ and EC₂₅. Data presented as percentages of mitoxantrone EC₅₀ values in each cell line, averages ± SD, n = 3. Statistical analysis by 1-way Anova, *p<0.05 for drug EC₅₀ values that were significantly lower than the corresponding Ad5 values. The <i>dl</i>312 (ΔE1A) non-replicating virus served as negative control. D) Graphic representation of combination indexes (CI) generated from synergy studies with mitoxantrone in combination with each replicating viral mutant at two constant ratios 0.5 and 2.5 viral particles per cell (ppc)/nM drug. Synergistic interactions are represented by CI≤0.9, antagonism by CI≥1.1 and additive effects by 0.9</p