Safe and Efficient Gene Therapy for Pyruvate Kinase Deficiency

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1 The Amerian Soiety of Gene & Cell Therapy original artile Safe and Effiient Gene Therapy for Pyruvate Kinase Defiieny Maria Garia-Gomez 1,2, Andrea Calabria 3, Maria Garia-Bravo 1,2, Fabrizio Benedienti 3, Penelope Kosinski 4, Sergio López-Manzaneda 1,2, Collin Hill 4, María del Mar Mañu-Pereira 5, Miguel A Martín 1,2, Israel Orman 1,2, Joan-LLuis Vives-Corrons 5, Charles Kung 4, Axel Shambah 6, Shengfang Jin 4, Juan A Bueren 1,2, Eugenio Montini 3, Susana Navarro 1,2 and Jose C Segovia 1,2 1 Hematopoieti Innovative Therapies Division, Centro de Investigaiones Energétias, Medioambientales y Tenológias (CIEMAT) - Centro de Investigaiones Biomédias en Red de Enfermedades Raras (CIBERER), Madrid, Spain; 2 Advaned Therapies Mixed Unit. Instituto de Investigaión Sanitaria-Fundaión Jiménez Díaz (IIS-FJD), Madrid, Spain; 3 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Sientifi Institute, Milan, Italy; 4 Agios Pharmaeutials, Cambridge, MA, USA; 5 Red Cell Pathology Laboratory. Hospital Clíni of Barelona Institut d Investigaions Biomèdiques August Pi i Sunyer, Barelona, Spain; 6 Institute of Experimental Hematology at Hannover Medial Hospital, Hannover, Germany Pyruvate kinase defiieny (PKD) is a monogeni metaboli disease aused by mutations in the PKLR gene that leads to hemolyti anemia of variable symptomatology and that an be fatal during the neonatal period. PKD reessive inheritane trait and its urative treatment by allogenei bone marrow transplantation provide an ideal senario for developing gene therapy approahes. Here, we provide a prelinial gene therapy for PKD based on a lentiviral vetor harboring the hpgk eukaryoti promoter that drives the expression of the PKLR DNA. This therapeuti vetor was used to transdue mouse PKD hematopoieti stem ells (HSCs) that were subsequently transplanted into myeloablated PKD mie. Etopi RPK expression normalized the erythroid ompartment orreting the hematologial phenotype and reverting organ pathology. Metabolomi studies demonstrated funtional orretion of the glyolyti pathway in RBCs derived from genetially orreted PKD HSCs, with no metaboli disturbanes in leukoytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoieti ells demonstrated no evidene of genotoxiity in any of the transplanted animals. Overall, our results undersore the therapeuti potential of the hpgk-orpk lentiviral vetor and provide high expetations toward the gene therapy of PKD and other erythroid metaboli geneti disorders. Reeived 2 August 215; aepted 25 Marh 216; advane online publiation 14 June 216. doi:1.138/mt INTRODUCTION Among many other hereditary enzymati defets affeting the erythroytes, pyruvate kinase defiieny (PKD) is the most frequent one ausing hroni nonspheroyti hemolyti anemia. 1 Onset and severity of PKD are very variable and range from mild to severe neonatal anemia, beoming fatal during the hildhood in the most severe ases. 2 Growth retardation, hydrops fetalis, and death during the neonatal period have also been reported with low frequeny. 3 PKD prevalene has been estimated at 1:2, in the general Cauasian population 4 and, so far, more than 195 different mutations in the PKLR gene have been identified ( lovd.nl/pklr). Allogenei bone marrow transplantation has been suessfully used to ure severe PKD patients, 5 but the low availability of histoompatible donors and the serious ompliations assoiated with the bone marrow transplantation of these patients (i.e., graft versus host disease, opportunisti infetions, et.) make periodi blood transfusions and splenetomy the main therapeuti options for most of the severe forms of PKD, 6 dramatially inreasing patient morbidity and mortality. 7 The limited effiay and side effets of the therapeuti options for severe PKD patients and its reessive inheritane trait make PKD a suitable disease to be treated by gene therapy. PKD is aused by defets in the pyruvate kinase (PK) enzyme 6 that atalyzes the last ATP-generating reation of the glyolysis pathway in all ells. In mature erythroytes, PK beomes essential as RBCs only express the R-type-speifi isoform (RPK) 8 due to the regulation of the erythroid-speifi alternative promoter of the PKLR lous. 9 Thus, any loss of RPK ativity impairs RBC metabolism and lifespan, 6 leading to hroni nonspheroyti hemolyti anemia. Gene therapy for monogeni diseases, partiularly those affeting the hematopoieti system, has provided onvining evidene that geneti orretion of autologous hematopoieti stem ells (HSCs) is an alternative therapeuti option to allogenei HSCT, avoiding its major ompliations Geneti orretion for diseases affeting the erythroyte suh as β-thalassemia and sikle ell disease, have been addressed in animal models 15,16 and also in humans. 11 However, gene therapy approahes for inherited erythroid metaboli defiienies suh as PKD are still limited. The feasibility of HSC gene therapy for PKD has been demonstrated both in mouse 17,18 and in dog RPK-defiient experimental models, 19 showing that donor himerism and transdution levels are Correspondene: Jose C Segovia, Hematopoieti Innovative Therapies Division, Centro de Investigaiones Energétias, Medioambientales y Tenológias (CIEMAT) - Centro de Investigaiones Biomédias en Red de Enfermedades Raras (CIBERER), CIEMAT (Edifiio 7), Av. Complutense, 4, 284 Madrid, Spain. j.segovia@iemat.es Moleular Therapy vol. 24 no. 7, jul

2 Gene Therapy for Pyruvate Kinase Defiieny The Amerian Soiety of Gene & Cell Therapy key points to reah an effiient orretion of the hemolyti phenotype 2 given the lak of seletive advantage of donor gene-orreted HSCs. Our previous work with a PKD mouse model demonstrated that retrovirally-derived human RPK expression was apable of fully orreting PKD phenotype when over 25% genetially orreted ells were transplanted. 18 A similar therapeuti threshold of orreted ells was reently reported in one PKD Basenji dog infused with foamy vetor-orreted and in vivo expanded HSCs. 19 High levels of transgene expression an be ahieved when γ-retroviral (γ-rv) vetors are used due to the fat that the therapeuti transgene expression is regulated by their long terminal repeats (LTR) sequenes. However, the first linial trials based on this type of vetors raised safety onerns, as several patients developed unexpeted leukemias. 21 The strong promoter ativity of LTR sequenes ould affet the regulation of surrounding genes, either by ativation of proto-onogene promoters or by inhibition of tumor suppressor genes, leading to insertional mutagenesis These findings highlighted the need to use safer and more effiient vetors than γ-retroviral vetors for the PKD gene therapy. Here, we provide an RPK lentiviral vetor (LV) for the geneti orretion of PKD. Geneti modifiation of murine PKD-HSCs with this vetor was able to effiiently orret the hemolyti phenotype and the RBC metabolite profile in transplanted PKD mie. Remarkably, no evidene of metaboli disturbanes in leukoytes and genotoxiity derived from the vetor integration were observed, supporting the therapeuti potential of the PGK-oRPK LV vetor. Overall, our prelinial results provide enouraging evidene of the feasibility of gene therapy for PKD with a LV designed for linial appliation. RESULTS PGK-oRPK therapeuti lentiviral vetor leads to a stable and long-term orretion of the anemi phenotype in genetially orreted PKD mie In vivo effiay of the PGK-oRPK LV (Supplementary Figure S1a) was assayed by transdution and transplantation of lineage depleted BM ells (Lin - ells) from PKD mie (Supplementary Figure S1b). Lethally irradiated PKD mie transplanted with defiient ells transdued with the orpk LV showed a signifiant improvement in all tested blood erythroid parameters when ompared to nontransplanted PKD littermates or to mie transplanted with ells transdued with an enhaned green fluoresent protein (EGFP) LV (Figure 1 and Supplementary Table S1). RBC ounts inreased as soon as 4 days post-transplantation (Figure 1a) and onstitutive retiuloytosis, whih is one of the most ommon signs of PKD, was signifiantly reverted in mie arrying the PGK-oRPK transgene, reahing levels lose to those observed in healthy ontrols for at least 9 months after transplant (Figure 1b). On the ontrary, PKD animals transplanted with EGFP LV-transdued ells showed anemia and remarkable retiuloytosis in parallel with PKD mie at all time-points analyzed. Hemoglobin levels, hematorit index, mean orpusular volume, and mean orpusular hemoglobin values were also orreted in mie transplanted with lentivirally orreted ells when ompared to nontransplanted PKD littermates (Supplementary Table S1). This hematologial orretion was ahieved with ± 4.45% of donor himerism and transdution effiaies ranging from 6 to 9% (Supplementary Table S2). Transdued ells showed on average 1.65 ±.8 integrated vetor opies per ell (VCN), indiating that PGK-oRPK LV-vetor provided enough human RPK transgeni expression to revert the hemolyti anemia. Remarkably, the expression of the orpk transgene led to an extension of erythroid ell half-life when ompared to nontransplanted PKD mie (Figure 1,d). On average, PKD mie showed a RBC half-life of 19 days, while in genetially orreted mie this was extended to 25 days (6 days extension) reahing values lose to wild-type RBC half-life (Figure 1d). Thus, RBCs of orpk-expressing mie displayed intermediate survival kinetis between healthy and defiient ontrol mie (Figure 1), most likely beause full himerism was not ahieved in these animals (Supplementary Table S2). Nine months after transplant hematopoieti progenitors from primary reipients were transplanted into seondary reipients that maintained engraftment levels (62.89 ± 5.61%) and viral opy number (VCN) (1.44 ±.8 opies) (Supplementary Table S2). Seondary transplanted reipients showed a multi-lineage hematopoieti reonstitution up to 5 months post-transplantation (Supplementary Figure S2) and a signifiant improvement in all PB erythroid parameters (Figure 2 and Supplementary Table S1). In addition, proviral integrations were deteted in differently ommitted hematopoieti progenitors (Supplementary Figure S3a,b) and its number remained onstant over time (Supplementary Figure S3) demonstrating the stability of the geneti orretion and highlighting the safety of the PGK-oRPK LV. Lentiviral-derived RPK expression normalizes erythroid differentiation and allows the prodution of funtional mature erythroytes PKD mie show a harateristi expansion of the erythroid ompartment aused by the ompensatory erythropoiesis mehanism. 26 The study of the erythroid differentiation pattern in transplanted mie indiated that the etopi RPK expression reverted this mehanism (Figure 3a,b). PKD and EGFP-expressing mie showed a predominane of immature erythroid preursors (subpopulation I: proerythroblasts and subpopulation II: basophili erythroblasts) in BM and spleen, and a remarkable fall in late erythroid ells (population IV: retiuloytes and mature erythroytes), while mie transplanted with ells transdued with the orpk LV showed a signifiant redution of immature erythroid preursors (subpopulations I and II) in BM and spleen, and a signifiant inrease of the latest erythroid ompartment (subpopulation IV) equivalent to healthy mie (Figure 3a,b). In addition, unlike PKD and EGFP-expressing mie, those arrying the orpk transgene showed a signifiant redution of erythropoietin (Epo) levels in plasma (Figure 3). The normalization of the erythropoiesis in PKD mie treated with the therapeuti vetor was aompanied by a redution of the spleni number of progenitors ontent to normal levels (Supplementary Figure S4a), although no hanges in the BM olony forming units (CFU) ontent were noted (Supplementary Figure S4b). Transplantation of ells transdued with the orpk lentiviral vetor reverts extramedullar erythropoiesis and organ pathology Due to the ative destrution of RPK defiient erythroytes, PKD and EGFP-expressing mie displayed aute splenomegaly with vol. 24 no. 7 jul. 216

3 The Amerian Soiety of Gene & Cell Therapy Gene Therapy for Pyruvate Kinase Defiieny a 12 b 45 RBC (1 9 /ml) 6 % Retiuloytes Days post-transplantation Days post-transplantation Day Day 2 Day 8 Day 12 Day % % % % % Healthy % % % % % Biotin PKD % % % % % CoRPK Ter119 d 1 8 % Biotin + RBC Days postinjetion Figure 1 Corretion of pyruvate kinase defiieny (PKD) phenotype in peripheral blood of primary reipients after geneti orretion. (a) RBCs and (b) retiuloyte levels in healthy (blak bar, n = 5) and PKD anemi mie (gray bar, n = 6), and PKD anemi mie that were tranplanted with EGFP (white bar, n = 9) or orpk-transdued ells (srathed bar, n = 17). Data are represented as the average ± SEM and were analyzed by nonparametri Kruskal-Wallis test. () Flow ytometry strategy used to detet the biotin-labeled RBCs throughout the time and (d) RBC survival kinetis in healthy (blak dots, n = 2), anemi (white dots, n = 2) and genetially orreted mie (disontinuous line, n = 4). Data are represented as the average ± standard error of the mean and were analyzed by two-way analysis of variane test. Healthy, nontransplanted ontrol mie; PKD, nontransplanted PKD mie; orpk, PKD mie expressing the therapeuti transgene. an inrease of spleen weight and size of over 2% when ompared to healthy ontrols (Figure 4a,b). A disorganized struture of the spleni tissue and the expansion of the spleen red pulp were also observed in these animals, indiating an intense extramedullar erythropoiesis also supported by the presene of erythroid ell lusters in PKD and EGFP-expressing liver setions (Figure 4). Remarkably, the etopi expression of orpk transgene ompletely reverted spleen and liver pathology in genetially Moleular Therapy vol. 24 no. 7 jul

4 Gene Therapy for Pyruvate Kinase Defiieny The Amerian Soiety of Gene & Cell Therapy a Healthy mie PKD 2nd orpk 1 μm 1 μm 1 μm b Side satter % 28.3% 9.9% Erythroid RNA + fration RBC (1 9 /ml) d % Retiuloytes Days post-transplantation 3 14 Days post-transplantation Figure 2 Pyruvate kinase defiieny phenotype orretion in seondary transplanted mie. (a) Brilliant Cresyl blue staining of blood smears from nontransplanted mie and seondary (2nd orpk) reipients to identify retiuloyte population (in blue). (b) Flow ytometry analysis of retiuloyte levels in peripheral blood. () RBCs and (d) retiuloyte perentage in seondary transplanted mie expressing the orpk transgene (srathed bar, n = 4), in healthy mie (blak bar, n = 3) and in anemi ontrol mie (gray bar, n = 3). Data are represented the average ± standard error of the mean and were analyzed by nonparametri two-tailed Mann-Whitney test. orreted mie, reduing the RBC aumulations and normalizing spleen histologial struture and size (Figure 4). Additionally, histologial studies revealed the total absene of iron deposits in the liver of genetially orreted mie, whereas PKD mie either from the nontransplanted group or the group transplanted with HSCs transdued with the EGFP-arrying vetor displayed an intense iron overload due to the ontinuous hemolyti proess (Figure 4). Overall, the transplant of genetially orreted HSCs in PKD mie restored the normal status of the erythropoiesis and all the seondary effets aused by the hemolyti anemia. PGK-oRPK LV-derived expression restores the glyolysis pathway in RBCs without modifying the WBC metaboli balane Next, we performed an extensive metabolomi analysis of all transplanted and ontrol mie to study the funtional orretion of RPK enzymati ativity. Following an untargeted profiling strategy, we observed signifiant hanges of glyolyti intermediates in RBCs among the different groups, identifying three broad lusters of metabolite patterns with distint trends (Figure 5a). RBCs from orpk-expressing mie showed an inrease of metabolites from luster 1 similar to healthy ontrols but different from transplanted mie arrying the EGFP transgene. Likewise, luster 3 refleted a redued metabolite trend in genetially orreted mie similar to wild-type mie and different from EGFP-expressing mie. Nevertheless, luster 2 from assay 1 showed no differenes of metabolite profile between transplanted mouse groups (EGFP and orpk expressing mie) (Figure 5a). The untargeted metaboli profiling also showed that the geneti modifiation was apable of modifying some important glyolyti intermediates, ahieving an inrease in ATP (Figure 5b), pyruvate (Figure 5d) and derease in ADP levels (Figure 5) in erythroytes isolated from mie transplanted with PGK-oRPK LV-transdued HSCs. Considering these metaboli trends, we then analyzed other metabolites loated loser to the PK-atalyzed reation using a targeted profiling approah. Levels of the diret PK substrate phosphoenolpyruvate (Figure 5e) and the 3-phosphoglyerate (3-PG) (Figure 5f), loated upstream of the PK-atalyzed reation, approahed to vol. 24 no. 7 jul. 216

5 The Amerian Soiety of Gene & Cell Therapy Gene Therapy for Pyruvate Kinase Defiieny a 5 Healthy (n = 2) PKD (n = 3) Bone marrow 6 Spleen % Ter 119 vs CD71 ells EGFP (n = 2) 4 orpk (n = 9) I II III IV I II III Erythroid subpopulations IV b Healthy mie PKD ,3 CD71 orpk 1 2 EGFP 1 2 Epo (pg/ml) Healthy PKD EGFP orpk Ter119 Figure 3 Normalization of the erythroid differentiation pattern in genetially orreted mie. (a) Perentages of the different erythroid subpopulation in bone marrow and spleen at 14 days after transplant. (b) Representative dot plots of the flow ytometry strategy used. The expression intensity of the CD71 and Ter119 markers allows for identifying four erythroid subpopulations: population I: early proerythroblasts (Ter119 med CD71 high ), population II: basophili erythroblasts (Ter119 high CD71 high ), population III: late basophili and polyhromatophili erythroblasts (Ter119 high CD71 med ), and population IV: orthohromatophili erythroblasts, retiuloytes and mature erythroid ells (Ter119 high CD71 low ). () Plasma Epo levels measured by enzyme-linked immunosorbent assay (ELISA) in nontransplanted and transplanted mie. Dots represent values of individual mie. Lines represent average ± standard error of the mean and were analyzed by nonparametri Kruskal-Wallis test. Healthy, nontransplanted ontrol mie; PKD, nontransplanted PKD mie; EGFP, PKD mie expressing the EGFP transgene; orpk, PKD mie expressing the therapeuti transgene. PKD, pyruvate kinase defiieny. that of healthy ontrol mie. Defiient erythroytes expressing the orpk transgene also produed an inrease of D-latate (Figure 5g), the final produt of the anaerobi glyolysis, when ompared to PKD and EGFP expressing mie. To test whether the ompensation in the glyolyti metabolites was a result of the normalization in PK ativity in mature erythroytes, we measured the ativity of this enzyme and normalized it in relation to the Hexokinase (HK) ativity in order to avoid the influene of high amounts of retiuloytes in the defiient animals. RBCs were purified through a ellulose olumn to prevent leukoyte PK ativity ontamination. A omplete ompensation of PK ativity was observed in the animals expressing the orpk that reahed ratios similar to those obtained from wild-type healthy animals and from a normal healthy blood donor volunteer (Supplementary Figure S5). Prinipal omponent analysis showed that metabolite pattern of RBCs was different depending on the group and markedly different to WBC profile (Figure 6a). On the ontrary, WBC subgroups lustered together with very little differene among groups, indiating no hanges in the metaboli balane of leukoytes when expressing the etopi orpk (Figure 6a). Additionally, speifi metabolite hanges observed in the RBC untargeted profiling were not present in WBCs (Figure 6b d). Moleular Therapy vol. 24 no. 7 jul

6 Gene Therapy for Pyruvate Kinase Defiieny The Amerian Soiety of Gene & Cell Therapy a Healthy PKD EGFP orpk Spleen Liver Liver (Fe) Healthy mie 1 μm 1 μm 1 μm Healthy PKD 2nd orpk PKD 1 μm 1 μm 1 μm b Spleen weight (mg) / total body weight (mg) Healthy PKD EGFP orpk 2nd orpk EGFP orpk 1 μm 1 μm 1 μm 1 μm 1 μm 1 μm Figure 4 Reversion of splenomegaly and organ pathology in genetially orreted mie at 14 days post-transplantation. (a) Pitures of representative spleens and (b) ratio of spleen weight to total body weight from primary and seondary transplanted pyruvate kinase defiieny (PKD) mie. Dots represent values of individual mie. Lines represent average ± standard error of the mean per group. Data were analyzed by nonparametri Kruskal-Wallis test. () Histologial study of spleen and liver from primary transplanted PKD mie. First and seond olumn show the representative histology setions of spleen and liver stained with hematoxylin-eosin and photographed using a 4 and 1 objetive, respetively, in a light mirosope. Arrows point to erythroid ell lusters indiative of extramedullary erythropoiesis. Third olumn shows Prussian blue staining (Fe) of liver setions to detet iron deposits indiated by arrowheads. Photographs were taken using a 2 objetive. Group legends as in Figure 3. 2nd orpk, seondary reipients. PGK-oRPK LV-transdued ells render polylonal hematopoieti reonstitution without evidene of vetor genotoxiity Integration profile of LVs arrying either the orpk or the EGFP transgene was analyzed in transplanted mie. Resulting from the genome-wide integration profile of LVs, eah insertion reates a unique geneti mark that an be used to trak the lonal behavior in individual transdued ells. Genomi DNA (gdna) was obtained from WBCs and from BM ells and from primary and seondary transplanted mie, as well as from transdued ell pools before transplant (Lin - ells). Linear amplifiation-mediated polymerase hain reation (LAM-PCR) (Supplementary Figures S6 and S7) was used to amplify vetor/genome juntions, and to identify vetor insertion sites (ISs). PCR produts were sequened by MiSeq Illumina platform and the obtained sequenes were mapped onto the mouse genome by bioinformatis pipeline and filtered for ollisions as desribed in Supplementary Methods (Supplementary Figure S8). Overall, we mapped 5,173,892 sequening reads on the transplanted mouse genome, resulting in 2,22 unique vetor integration sites. The genomi distribution of ISs from two independent experiments mathed the previously reported LV preferene for integration within transriptional units (partiularly within the first 5 Kb downstream of the transription start site) (Supplementary Figure S9a), showing no skewing toward any partiular hromosome in the mouse genome (Supplementary Figure S9b). Safety of the PGK-oRPK LV-based gene therapy was studied by lonal abundane estimations, alulating the perentage of sequene ount for eah IS (a lonal mark) with respet to the total number of sequenes of the dataset. Dot plots and heat map representations of the relative abundane of eah IS retrieved for eah mouse (Figure 7; Supplementary Figures S1 and S11) showed strong flutuations in lonal omposition of the different mie and of in vitro ultured Lin - ells. Also, it was possible to appreiate that for several samples, a small number of integrations ontributed to a large amount of sequene reads (Figure 7 and Supplementary Figure S1), revealing a polylonal pattern of repopulation of transdued HSCs. In addition, traked shared integration between primary mie arrying the therapeuti PGKoRPK LV and subsequently transplanted seondary mie showed no strong sharing of integrations between the groups, onfirming the absene of lonal dominane (Supplementary Figure S1). To determine whether hallmarks of insertional mutagenesis were present in transplanted mie, we assessed the ourrene of ommon insertion sites (CIS) similar to urrently ongoing LV-mediated linial trials. 1,11,13,14,27 CIS are insertional hotspots that may result from integration bias at the time of transdution or in vivo seletion of lones harboring vetor integrations that vol. 24 no. 7 jul. 216

7 The Amerian Soiety of Gene & Cell Therapy Gene Therapy for Pyruvate Kinase Defiieny a b e vs Healthy Assay 1 Assay 2 vs egfp vs orpk vs Healthy vs egfp vs orpk d 3 ATP Assay 1 Assay 2 ADP Assay 1 Assay 2 Pyruvate f g Healthy PKD Healthy PKD PEP 3-PG EGFP CoRPK D-LACTATE EGFP CoRPK. Assay 1 Assay 2. Healthy PKD EGFP CoRPK Figure 5 Metaboli profiling in RBC samples from mie transplanted with genetially modified ells. Analysis of signifiant metaboli profile hanges in healthy and transplanted mie by omparison to pyruvate kinase defiieny (PKD) animals in two independent experiments. (a) Complete RBC heat map obtained by untargeted profiling, where higher and lower metabolite levels are represented in red and blue respetively. Metabolites listed have at least one omparison that is signifiant using the following riteria: absolute fold hange >1.5; minimal signal >2,; adjusted P value <.1. Blak boxes highlight luster of metabolite hanges with distint profile among the groups. (b d) ATP, ADP, and pyruvate levels in RBCs, respetively, measured by untargeted profiling by omparison to PKD mie at 14 days after transplant. Assay 1: Healthy mie (blak bars) n = 1, PKD (gray bars) n = 1, hpgk-egfp (white bars) n = 2, hpgk-orpk (srathed bars) n = 3. Assay 2: Healthy mie n = 2, PKD n = 2, hpgk-egfp n = 6, hpgk-orpk n = 1. (e g) RBC targeted metaboli profiling of a seleted number of metabolites involved in the glyolyti pathway (phosphoenolpyruvate, 3-phosphoglyeri aid and D-lati aid, respetively) at 28 days post-transplantation. Dots represent values of individual mie. Lines represent average ± standard error of the mean and were analyzed by nonparametri Kruskal-Wallis test. Assay 2: Healthy mie n = 7, PKD n = 5, hpgk-egfp n = 3, hpgk-orpk n = 5. onfer growth advantage. 22,28 31 CIS were identified using an algorithm based on Abel and ols 32 and the Grubbs test for outliers, 27 finding no CIS and thus no alarming signs of genotoxiity by this readout. Moreover, gene ontology analysis revealed no skewing toward gene lasses involved in aner, ell proliferation or regulation of apoptosis in any of the integration datasets sorted by tissue-distribution, time point or abundane of repopulating hematopoieti ell lones (Supplementary Figure S12). These results suggest neutrality of vetor integration and demonstrate the safety of the PGK-oRPK LV in a prelinial setting. DISCUSSION Geneti orretion of erythroid metaboli diseases suh as Gluose-6-phosphate dehydrogenase (G6PD) defiieny and PKD has yielded promising results in animal models, 18,33 but the insertional onogeni risk observed in the first gene therapy trials using γ-rv vetors 21,24 made neessary to develop safer and more sophistiated tools to provide gene therapy a valid therapeuti option for PKD. Here, we show prelinial evidene that the PGKoRPK LV effiiently resues PKD pathology upon geneti orretion and transplantation of HSCs in defiient mie. Furthermore, the safety-optimized design of the therapeuti vetor leads to a safe geneti orretion without modifying metaboli energy balane in leukoytes or ausing side effets derived from vetor integration. Based on the effiay and safety properties of LVs urrently used in HSC gene therapy, we designed a self-inativating LV arrying a odon-optimized version of the human wild-type PKLR gene (orpk) that led to normal levels of all hematologial variables (RBCs, hemoglobin levels, hematorit index, mean orpusular volume, and mean orpusular hemoglobin) and rapidly restored RPK to normal funtioning in erythroid ells providing normal retiuloyte and inmature erythroide ell levels in gene therapy treated mie lose to healthy ontrols. The PGK-oRPK LV treatment also led to an inreased RBC survival, proportional Moleular Therapy vol. 24 no. 7 jul

8 Gene Therapy for Pyruvate Kinase Defiieny The Amerian Soiety of Gene & Cell Therapy a b 3 ATP 6 PCA3_ PCA1_ PCA2_ Assay 1 Assay 2 3, ADP d Pyruvate 2, 1, 5 25 Assay 1 Assay 2 Assay 1 Assay 2 Figure 6 Untargeted metaboli profiling in WBC samples from mie transplanted with genetially modified ells. (a) Prinipal omponent analysis of untargeted metabolite profile in RBCs (red dots) and WBCs (blue dots) in ontrol and transplanted mie. (b d) ATP, ADP, and pyruvate levels, respetively, in WBCs by omparison to pyruvate kinase defiieny (PKD) mie. Assay 1: healthy mie (blak bars) n = 1, PKD (gray bars) n = 1, hpgk-egfp (white bars) n = 2, hpgk-orpk (srathed bars) n = 3. Assay 2: healthy mie n = 2, PKD n = 2, hpgk-egfp n = 6, hpgk-orpk n = 1. Data represent the average ± standard error of the mean per group and were analyzed by nonparametri Kruskal-Wallis test. to the level of donor himerism deteted in genetially orreted mie. In PKD pathology, the hemolyti proess triggers the mehanism of ompensatory erythropoiesis to mitigate the quik loss of damaged RBCs, but this rapid development of new erythroytes turns out that it is ineffetive leading to an inreased apoptosis in the erythroid ompartment. 34 Some of the proposed explanations are membrane rigidity 35 and metaboli blok aused by glyolyti intermediates aumulation 6,36 and the potential impairment of oxidative stress response 36 as a onsequene of ATP depletion. The geneti orretion with the PGK-oRPK LV however, normalizes the erythroid differentiation pattern in BM and spleen beause the vetor-derived RPK expression leads to an improved metaboli status of the basophili erythroblasts, the stage in whih RPK expression starts to be predominant 37 and allows the proper differentiation into the next stage of erythroid maturation. The orretion of ineffetive erythropoiesis in transplanted mie suggests that the PGK promoter drives a stable and robust expression of the orpk transgene, able to resist the epigeneti hanges that erythroid progenitor ells undergo upon differentiation into mature RBCs. On top of that, the harateristi splenomegaly present both in patients 6 and in PKD mie 38 was also reverted after geneti orretion even with a moderate perentage of transdued ells (62% of CFU progenitors arrying the integrated provirus). Redution of extramedullary erythropoiesis and iron overload in the spleen and liver of transplanted mie, signifiant redution of plasma Epo levels and spleen CFU ontent normalization in orpk-expressing mie demonstrates an almost omplete reovery of the PKD pathology to the normal stage. Nevertheless, EGFP-expressing mie show a slight redution of spleen weight that may be related to the effets assoiated to the hematopoieti transplantation proedure, suh as the proliferative stage of the hematopoieti progenitors after the transplant. The PKD phenotype orretion provided by the developed therapeuti vetor was further onfirmed by metabolomi analysis, demonstrating that the metabolite profile of the orreted erythroytes is similar to the healthy one and different from PKD mie and from PKD mie transplanted with ells transdued with the EGFP expressing vetor. Although untargeted metabolite profiling shows luster similarities between groups of transplanted mie (luster 2, egfp and orpk), these similarities ould be attributed to the BM transplant proedure that would affet both groups, and no matter the PKD phenotype was orreted. Clusters 1 and 3 reveal an inrease and a derease metabolite trend, respetively, in genetially orreted mie and in healthy ontrols that is different from mie arrying the EGFP transgene. This inludes an inrease of pyruvate and ATP levels and a derease of ADP in RBCs that, in spite of not being signifiant, probably beause of the bulk metabolite analysis, suggests the orretion of the glyolyti pathway upon geneti orretion. Targeted metabolomi analysis showed a signifiant vol. 24 no. 7 jul. 216

9 The Amerian Soiety of Gene & Cell Therapy Gene Therapy for Pyruvate Kinase Defiieny a % IS Seq ount BM PB BM PB BM PB b % IS Seq ount orpk1 orpk2 orpk Lin BM PB BM PB BM PB BM PB BM PB BM PB BM PB BM PB BM PB BM PB orpk1 orpk2 orpk3 orpk4 orpk5 orpk6 orpk7 orpk8 orpk9 orpk1 99. % IS Seq ount PB BM orpk11 orpk12 orpk13 orpk14 orpk2.1 orpk2.2 orpk2.3 orpk2.4 Figure 7 Clonal abundane analysis of orpk-lv-transdued ells. Dots plot representation of lonal abundane of pooled integrations in eah mouse from assays 1 (a) and 2 (b,). The relative perentage (y-axis) for eah integration site is relative to the total number of sequenes reads obtained in eah dataset. BM, bone marrow; IS, integration site; PB, peripheral blood; orpk1-14, mie transplanted with hematopoieti ells transdued with the therapeuti vetor. orpk , seondary reipients transplanted with BM ells from orpk primary mie. orpk11 orpk12 orpk13 orpk14 orpk2.1 orpk2.2 orpk2.3 Primary reipients Seondary reipients Primary reipients Seondary reipients orpk2.4 normalization not only of relevant metabolites loated upstream of the RPK-atalyzed reation (phosphoenolpyruvate, 3-PG, and D-latate), but also intermediates of pentose phosphate pathway (not shown), pointing to a normal funtion of glyolysis pathway when RBCs express funtional RPKs. Moreover, the PK enzyme ativity, showed as the ratio PK/HK to ompensate the differenes in the amount of retiuloytes in healthy and diseased animals, learly points toward the omplete reovery of the PK funtion in the orreted erythroytes. Interestingly, the ratio obtained was also similar to the one obtained in blood from a normal healthy volunteer, suggesting physiologial levels of vetor-derived PK ativity and further demonstrating the apaity of the vetor to ompensate PKD. Remarkably, WBCs analyzed in parallel showed no alterations of the metaboli balane in leukoytes when RPK is etopially expressed under the ontrol of a ubiquitous promoter suh as PGK, ruling out a leukoyte metaboli advantage as a possible safety onern and reinforing the therapeuti potential of the PGK-oRPK LV. The multi-lineage reonstitution and the absene of any leukemi event or lonal expansion in seondary reipients demonstrate the long-term stability and safety of the PGK-oRPK LV-based protool that was further onfirmed through the analysis of vetor integration sites, an approah that is being inreasingly used to predit the risk of insertional onogenesis in HSC gene therapy linial trials and in prelinial studies. 1,11,13,14,22,27,28,3,31 The analysis relative to the abundane of speifi ell lones arrying the integrated vetor shows a limited number of vetor-marked lones repopulating the hematopoiesis in genetially orreted mie. Despite this polylonal reonstitution pattern, CIS analysis did not Moleular Therapy vol. 24 no. 7 jul

10 Gene Therapy for Pyruvate Kinase Defiieny The Amerian Soiety of Gene & Cell Therapy show any alarming sign of genotoxiity. Also, highly represented lones found at a ertain time-point after transplant in some mie were redued at longer times or absent in BM, indiating a high turnover and no lonal dominane of PGK-oRPK LV-orreted HSCs. Importantly, seondary transplanted mie showed integration sites found in primary reipients indiating that stem ells were marked, and in new integration sites not previously deteted in primary reipients, pointing to the healthy lonal dynamis of hematopoiesis after geneti orretion. These results suggest that the polylonal nature of reonstitution observed is likely a onsequene of the biology of hematopoieti reonstitution in mie. 39 In addition, gene ontology analysis revealed no skewing toward gene lasses involved in aner, ell proliferation or regulation of apoptosis. 4 Interestingly, the gene ontology study also showed that genes involved in atabolism were the ones most targeted by vetor integration. This trend is different to that observed in other studies direted to other gene defiienies, 41,42 and suggests that the viral integration pattern ould be biased by the geneti defiieny of the reipient animals. Overall, the analysis of the vetor integration pattern emphasizes the safety properties of the PGK-oRPK LV that provides PKD geneti orretion with no signs of genotoxiity. The development of gene therapy protools for PKD is hallenging and requires the preise balane between effiay and safety. Firstly, the lak of seletive advantage of orreted HSCs in erythroid metaboli defiienies suh as PKD 18 2 makes neessary the development of effiient vetors to ensure the expression of the transgene in mature affeted RBCs and high levels of himerism to ensure a high prodution of orreted erythroytes. Seondly, high levels of wild-type RPK expression would be desirable to inrease the expression of human monomers, inreasing the probability of aumulating funtional RPK tetramers, sine the formation of heterotetramers between vetor-derived funtional opies of PK and endogenous nonfuntional monomers might redue the effiay of gene therapy for PKD. The PGK-oRPK LV leads to a omplete phenotype orretion when transdution levels fell to 62% with an estimated 1.2 transgene opies per ell, onsistent with the VCN obtained in other prelinial studies, i.e., β-thalassemia using a LV. 41 Nonetheless, pathologial manifestations are usually observed in PKD patients when RPK ativity falls below 25% of the normal ativity, 43 and a similar level of orreted ells has been reported therapeuti in previous gene therapy protools both in mouse 18 and dog models. 19 Hene, a phenotype orretion may be expeted even with lower levels of himerism 18,19 and it is reasonable to expet a therapeuti benefit even if normal levels of RPK expression are not ahieved after geneti orretion. In this senario, the singlegene PGK-oRPK LV design is essential and the use of the human PGK eukaryoti promoter, urrently being used in a linial trial, 14 provides transgene stable expression 44 and has been proven to be a weak transativator 45. Taken together, these have likely ontributed to its therapeuti effiay and safety, together with the optimization of the transgene sequene to inrease expression 46 and the inlusion of the mutated Wpre sequene to inrease mrna stability. 47 Our PGK-oRPK LV-based protool would be able to orret the wide number of mutations desribed in PKD patients 1,2,4 6,48 inluding mutations in the regulatory sequenes of the gene. 49,5 Also, onsidering that PKLR null mutations are rare, 6 the probability of developing an immunologial response against the exogenous protein would be redued. Even so, a patient-speifi strategy based on the CRISPR/Cas9 orretion of the endogenous lous would prevent the risk of insertional mutagenesis. This tehnology is urrently being developed in our laboratory using CD34 + HSCs from PKD patients with promising results, albeit with effiienies very far from potentially appliable to the linial setting yet. Overall, our findings demonstrate for the first time the feasibility of treating metaboli inherited erythroid disease through the geneti modifiation of HSCs using a safety- engineered LV vetor. MATERIALS AND METHODS Animals. The AB55 reombinant ongeni mie (also reorded as PKD mie) arrying the 269T>A loss-of-funtion mutation in the Pklr gene, were used throughout. 26,51 AB55 mie were obtained from Emerillon Therapeutis (Montreal, Quebe, Canada) and maintained at the Animal Faility Laboratory of CIEMAT (registration number ES ) by inbreeding. C57BL/6 mie, and oasionally A/J mie, were used as healthy ontrols. Lentiviral vetors prodution and transplantation of murine HSCs. LVs bakbones were generated as shown in Supplementary Figure S1a and vetors stoks were produed as previously desribed. 52 Lineage negative ells (Lin - ) were obtained from the BM of 8 14-week-old male PKD mie, prestimulated with 1 ng/ml of reombinant human IL-11 (Peproteh EC, London, UK) and 1 ng/ml of reombinant murine stem ell fator (SCF) (R&D Systems, Minneapolis, MN) for 24 hours and then transdued with EGFP or orpk arrying LVs in two yles of transdution at multipliities of infetion (MOIs) of 1 1 vp/ell (Supplementary Figure S1b). Eah transdution was arried out for 24 hours overnight. Approximately, (assay 1) to (assay 2 and 3) transdued Lin - per mouse were intravenously injeted into 8 14-week-old female PKD mie previously irradiated with a lethal dose of 9.5 Gy, split into two doses of 4.75 Gy 24 hours apart using a Philips MG324 X-ray instrument (Philips, Hamburg, Germany). Transplanted mie were monitored for 5 to 9 months and total BM was harvested at the end-point of the experiment to perform seondary transplants (4 1 6 ells/mouse). Hematologial variables and in vivo RBC survival. Mouse blood was olleted from tail veins and analyzed in an automated blood ell analyzer (Abaus Junior vet, CMV Analitia, Spain). Retiuloyte perentage was determined in nonoagulated blood by staining with.5 μg/ml of Aridine Orange (Invitrogen Life Tehnologies, Grand Island, NY) and analyzed in an EPICS XL flow ytometer (Bekman Coulter Eletronis, Hialeah, FL). Additionally, retiuloytes were identified in blood smears by Brilliant Cresyl Blue staining (Reagena, Toivala, Finland) and Giemsa ounterstaining, and analyzed in a Leia Mirosystems mirosope. In vivo staining of RBCs was arried out following a previously published protool of in vivo biotin staining. 38,53 Strutural and histologial studies. Spleen and liver were studied following onventional histologial methods (see Supplementary Information). Colony forming ell (CFC) assays, erythroid differentiation, and plasma erythropoietin (EPO) levels. CFC assays were performed following manufaturer s reommendations (Stem Cell Tehnologies, Vanouver, Canada). Erythroid differentiation was studied as desribed elsewhere 54 and Epo onentration was quantified using the Quantikine Mouse Epo immunoassay kit (MEPB R&D Systems). Provirus quantifiation and himerism. Detetion and quantifiation of integrated provirus per ell was aomplished by multiplex qpcr following a previously desribed strategy. 55 Additionally, olonies were singly piked to evaluate the VCN and the perentage of transdution. Chimerism levels were determined by deteting the presene of the Y hromosome as desribed elsewhere vol. 24 no. 7 jul. 216

11 The Amerian Soiety of Gene & Cell Therapy Gene Therapy for Pyruvate Kinase Defiieny Metabolomi studies. RBCs and WBCs were subjeted to metabolomis studies. 57 RBCs were obtained from the whole blood after 2,5 rpm/1 minute entrifugation to remove the plasma and WBC interfae. WBCs were obtained by erythroyte lysis and entrifugation of PB. Pellets were stored at 8 C. Metabolite extration was arried out by adding 5 volumes of 7 C 7% ethanol to one volume of ell pellet, and entrifugation at 3, rpm to pellet the preipitated protein. Thirty miroliters of supernatant were transferred to a 96-well plate and dried under nitrogen. Samples were then resuspended in 3 μl of water to allow for metabolite profiling by mass spetrometry. Two approahes were used to ollet metabolite profiles from the isolated blood omponents: (i) a targeted triple-quadruple-liquid hromatography mass spetrophotometry (LCMS) approah for the analysis of glyolyti and triarboxyli aid yle omponents, and (ii) an untargeted diret injetion approah oupled to a highmass-resolution Q-ToF. The targeted approah allowed for diret analysis of upstream/downstream metabolites from the pyruvate kinase enzyme, while the untargeted approah allowed for unbiased analysis of distal metaboli pathways. General strategy for integration site analysis. The integration site analysis was performed aording to the flowhart shown in Supplementary Figure S8 that inludes four main maro-ativities going from wet sample proessing to bioinformatis: 1. Wet lab proedures: LAM-PCRs and next-generation sequening. 2. Next-generation sequening data proessing: aquiring as input sequening reads from next-generation sequening platforms and getting as output the list of integration sites. 3. Data quality proessing: performing three sequential ativities to improve data quality. Eah ativity generates the input for distint proesses in step IS-driven biologial analysis: performing inferenes on safety and effiay of gene therapy. See also extended version Supplementary Text for further details. Abundane analysis. Clonality assessment was omputed from the matrix files leaned by ontaminations (also alled ollisions). We used the three groups of mie independently (orpk assay 1, EGFP assay 2, and orpk assay 2. See Supplementary Information). For eah group, the abundane value was obtained as the perentage for eah IS of the sequene reads over the total reads of the group (Figure 7 and Supplementary Figure S11). All values were obtained using Exel and then plotted the results using Prism. Exel files with the abundane representation as heat map are also available (Supplementary Figure S1). Statistial analysis. Data from all experiments were represented as the average ± standard error of the mean. Two different nonparametri statistial tests were applied depending on the number of groups with no assumption of normal distribution of the data. When three or more experimental groups were ompared, we used the Kruskal-Wallis test (P <.5; P <.1; P <.1), whereas two-tailed Mann-Whitney test was applied when only two groups were ompared. RBC half-life studies were analyzed by two-way analysis of variane test (P <.5; P <.1; P <.1). Comparisons were arried out between transplanted mie and PKD littermates, and all statistial analyses were performed using GraphPad Prism software, version 5.a (GraphPad Software, San Diego, CA). Study approval. All experimental proedures were arried out aording to European and Spanish laws and regulations (European onvention ETS 1 2 3, regarding the use and protetion of vertebrate mammals used in experimentation and other sientifi purposes and Spanish Law 32/27, R.D. 121/25 and RD 53/213 regarding the protetion and use of animals in sientifi researh). Proedures involving Genetially Modified Organism were approved by Spanish Biohazard Commission (Registration Number A/ES/4/I-4). SUPPLEMENTARY MATERIAL Table S1. Hematologial variables reorded 14 days post-transplantation in peripheral blood. Table S2. Relevant moleular parameters in mie transplanted with genetially modified ells. Figure S1. Experiment design. Figure S2. Multi-lineage hematopoieti reonstitution in seondary transplanted mie. Figure S3. Quantifiation of proviral integrations. Figure S4. Hematopoieti progenitor assays in ontrol mie and transplanted mie with transdued ells Figure S5. Pyruvate Kinase and Hexokinase ativity in mie transplanted with transdued ells. Figure S6. Gel image of LAM-PCR produts generated with Tsp59I enzyme for samples harvested from all mie at different time points and tissues as Fig. S8 shows. Figure S7. Gel image of LAM-PCR produts generated with HpyCH4IV5 enzyme for samples harvested from all mie at different time points and tissues as Fig. S8 shows. Figure S8. General sheme of the analysis of integration site mapping performed in mie transplanted with genetially modified hematopoieti progenitors. Figure S9. Distribution of LV integrations along the genome of transplanted mie. Figure S1. Traked shared integrations between primary and seondary reipient mie arrying the therapeuti PGK-oRPK LV vetor. Figure S11. Clonal abundane analysis of EGFP-LV transdued ells. Figure S12. LV genomi integration profile. Supplementary Methods. Supplementary Information. Supplementary Text. ACKNOWLEDGMENTS The authors would like to thank Aurora de la Cal and Sergio Losada for their ollaboration in the administrative work; Jesús Martínez and Edilia de Almeida for their areful maintenane of the animals, and Norman Feltz for reading and orreting the manusript for style and grammar. This work was supported by the following grants: Direión General de Investigaión de la Comunidad de Madrid (CellCAM; Ref S21/ BMD-242), Fondo de Investigaiones Sanitarias, Instituto de Salud Carlos III (RETICS-RD12/19/23) and MINECO (SAF R and SAF ). The authors delare no onflit of interest. REFERENCES 1. Zanella, A, Bianhi, P and Fermo, E (27). Pyruvate kinase defiieny. Haematologia 92: Pissard, S, Max-Audit, I, Skopinski, L, Vasson, A, Vivien, P, Bimet, C et al. (26). Pyruvate kinase defiieny in Frane: a 3-year study reveals 27 new mutations. Br J Haematol 133: Gilsanz, F, Vega, MA, Gómez-Castillo, E, Ruiz-Balda, JA and Omeñaa, F (1993). Fetal anaemia due to pyruvate kinase defiieny. 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The L- and R-type isozymes of rat pyruvate kinase are produed from a single gene by use of different promoters. J Biol Chem 262: Cartier, N, Haein-Bey-Abina, S, Bartholomae, CC, Veres, G, Shmidt, M, Kutshera, I et al. (29). Hematopoieti stem ell gene therapy with a lentiviral vetor in X-linked adrenoleukodystrophy. Siene 326: Cavazzana-Calvo, M, Payen, E, Negre, O, Wang, G, Hehir, K, Fusil, F et al. (21). Transfusion independene and HMGA2 ativation after gene therapy of human β-thalassaemia. Nature 467: Moleular Therapy vol. 24 no. 7 jul