Human Phosphoglycerate Kinase Research Articles

Metastable States in the Hinge-Bending Landscape of an Enzyme in an Atomistic Cytoplasm Simulation.

Many enzymes undergo major conformational changes to function in cells, particularly when they bind to more than one substrate. We quantify the large-amplitude hinge-bending landscape of human phosphoglycerate kinase (PGK) in a human cytoplasm. Approximately 70 μs of all-atom simulations, upon coarse graining, reveal three metastable states of PGK with different hinge angle distributions and additional substates. The "open" state was more populated than the "semi-open" or "closed" states. In addition to free energies and barriers within the landscape, we characterized the average transition state passage time of ≈0.3 μs and reversible substrate and product binding. Human PGK in a dilute solution simulation shows a transition directly from the open to closed states, in agreement with previous SAXS experiments, suggesting that the cell-like model environment promotes stability of the human PGK semi-open state. Yeast PGK also sampled three metastable states within the cytoplasm model, with the closed state favored in our simulation.

Enhanced expression of the human Survival motor neuron 1 gene from a codon-optimised cDNA transgene in vitro and in vivo.

Spinal muscular atrophy (SMA) is a neuromuscular disease particularly characterised by degeneration of ventral motor neurons. Survival motor neuron (SMN) 1 gene mutations cause SMA, and gene addition strategies to replace the faulty SMN1 copy are a therapeutic option. We have developed a novel, codon-optimised hSMN1 transgene and produced integration-proficient and integration-deficient lentiviral vectors with cytomegalovirus (CMV), human synapsin (hSYN) or human phosphoglycerate kinase (hPGK) promoters to determine the optimal expression cassette configuration. Integrating, CMV-driven and codon-optimised hSMN1 lentiviral vectors resulted in the highest production of functional SMN protein in vitro. Integration-deficient lentiviral vectors also led to significant expression of the optimised transgene and are expected to be safer than integrating vectors. Lentiviral delivery in culture led to activation of the DNA damage response, in particular elevating levels of phosphorylated ataxia telangiectasia mutated (pATM) and γH2AX, but the optimised hSMN1 transgene showed some protective effects. Neonatal delivery of adeno-associated viral vector (AAV9) vector encoding the optimised transgene to the Smn2B/- mouse model of SMA resulted in a significant increase of SMN protein levels in liver and spinal cord. This work shows the potential of a novel codon-optimised hSMN1 transgene as a therapeutic strategy for SMA.

A novel hybrid promoter capable of continuously producing proteins in high yield

The establishment of cell lines with a high protein production is the most crucial objective in the field of biopharmaceuticals. To this end, efforts have been made to increase transgene expression through promoter improvement, but the efficiency or stability of protein production was insufficient for use in commercial production. Here, we developed a novel strategy to increase the efficiency and stability of protein production by hybridizing a promoter that exhibits higher expression levels at the transient level with a promoter that exhibits higher stability at the stable level. Expression levels of transgenes by each promoter were measured at transient and stable levels for five single promoters: Rous sarcoma virus (RSV), cytomegalovirus (CMV), human phosphoglycerate kinase (hPGK), simian virus 40 (SV40), and zebrafish ubiquitin B (Ubb). The hPGK promoter enabled high-yield transgene expression at transient levels and the SV40 promoter enabled sustained expression at stable levels. Therefore, hPGK and SV40 promoters were selected as candidates for establishing hybrid promoters and two hybrid promoters were constructed; one hybrid promoter in which the SV40 promoter is added before the hPGK promoter (a.k.a. SKYI) and the other hybrid promoter in which the SV40 promoter is added after the hPGK promoter (a.k.a. SKYII). Of the two hybrid promoters, the hybrid promoter SKYII promoted high-yield transgene expression at both transient and stable levels compared to single hPGK and SV40. Together, our findings open new doors in the field of biopharmaceuticals by presenting a novel promoter platform that can be used for high-yield and sustained protein production.

Molecular mechanism of glycolytic flux control intrinsic to human phosphoglycerate kinase

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.

Improved Gene Therapy for Metachromatic Leukodystrophy

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease (LSD) characterized by a decreased Arylsulfatatse A (ARSA) enzymatic activity. The most common form, late infantile MLD, universally results in rapid loss of neurologic function in early childhood. Ex-vivo hematopoietic stem cell (HSC) gene therapy using a lentiviral vector (LV) can improve clinical outcomes by supplying a functional copy of the ARSA cDNA (Biffi A, et al, Science 2013). Unfortunately, this approach is only successful in pre- and minimally symptomatic individuals and only a small subset of individuals are diagnosed during the limited therapeutic window. As such, the development of additional approaches targeting early symptomatic individuals are critically needed.The only clinical vector (CV) approved to treat MLD patients, PawMut6, includes the human ARSA cDNA gene under the control of the human Phosphoglycerate Kinase (PGK) promoter and includes, in the integrating transcriptional unit, the viral sequences Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence to increase titer and mRNA translation (Biffi A, et al, Science 2013). To increase expression of ARSA cDNA at single integration level, we generated several LVs that include the ARSA gene with a variety of insulators to optimize ARSA expression and enhance safety in transduced cell lines. We placed the ARSA cDNA under the control of the human Elongation Factor 1 alpha (EF1-alpha) promoter, which has been shown to promote higher transcription rates in different cell lines, compared to human PGK as shown by Jane Yuxia Qin, PLos One 2010. Our constructs carry versions of the ARSA gene with and without the 5' and 3' untranslated regions (UTR+ or UTR-) and a Traceable Codon Optimized (TCO) modified sequence to distinguish the transgene from the endogenous ARSA. An ankyrin or foamy insulator have been incorporated to minimize genotoxicity caused by integration events. The WPRE has been proven to enhance the performance of viral vectors. However, to prevent WPRE integration in the host genome, we placed it directly after the 3'-self inactivating LTR (SIN-LTR) together with a strong bovine growth hormone polyA signal (for sequence termination) (BGHpA), as shown by Breda L. et al, Mol Ther 2021.We compared the ARSA activity (normalized to vector copy number (VCN)) of our constructs to that of PawMut6, the LV currently used in clinical trial, on MLD primary patient fibroblast cultures. Our top performing vectors, TCO-EAAWP-UTR +, TCO-EAFWP-UTR - and TCO-AEAFWP-UTR - showed 2X, 10X and 4X more ARSA activity, respectively, compared to that generated using PawMut6. We also detected a superior ability of our vectors to secrete functional ARSA enzyme into the culture media of transduced primary MLD patient fibroblast cells, which is a critical modality for transfer of functional ARSA from microglia to oligodendrocytes. Extracellular vesicle isolation, purification, and immunoblot analysis has demonstrated small vesicle secretion is the primary modality by which ARSA is secreted, having significant implication for how we approach treatment of MLD. In parallel experiments on murine HSC, the TCO-AEAFWP-UTR - vector reproduced similar results, with about 4x more ARSA activity. To exclude potential toxicity, we performed bone marrow transplants on WT animals with HSCs transduced at up to 13 copies per genome. Mice transplanted with high VCN transduced bone marrow did not show signs of bone marrow failure or distress; more extensive evaluation of these animal models is ongoing. Clonogenic assays and secondary transplants are in progress. Upon completion of the in-vivo studies in WT mice, at least two of our best vector candidates will be utilized on a MLD mouse model (ARSA-KO) that we generated using CRISPR-Cas9. Analysis will include pathological sections of the CNS, brain lysate collection and sulfatase activity assays. Our studies are currently focused on completing in-vivo validation and toxicity assays to move our best vector to the pre-clinical and IND application. The accumulated data on our novel vectors imply new mechanistic considerations for treatment of MLD and demonstrate utility as a strong approach for treating early symptomatic patients. DisclosuresVanderver: Homology: Research Funding; Takeda: Research Funding; Ionis and Illumina Inc: Research Funding; Biogen: Research Funding; Eli Lily and Company: Research Funding; Orchard Therapeutics: Research Funding; Gilead Sciences Inc: Research Funding. Adang: MEGMA: Consultancy; Orchard Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Rivella: Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Forma Theraputics: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients.Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction.In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. DisclosuresBianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

Identification of a novel variant in phosphoglycerate kinase-1 (PGK1) in an African-American child (PGK1 Detroit)

The human phosphoglycerate kinase-1 enzyme is the first of two energy generating steps in the glycolysis. Since its discovery in 1968, many pathologically mutated forms of PGK1 have been described. PGK1 is expressed in all tissues. The clinical manifestations of PGK1 deficiency are some combination of anemia, central nervous system and/or musculoskeletal manifestations. We describe a case of PGK1 in an African-American child, which to our knowledge, has never been described to date. The manifestations of PGK1-Detroit (c.1105A > C (p.Thr369Pro)) include hematologic and central nervous manifestations.

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

AbstractHematopoietic 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.

Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp−/−Upp1−/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2–3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp−/−Upp1−/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

Polyclonal origin of parathyroid tumors is common and is associated with multiple gland disease in primary hyperparathyroidism

Parathyroid tumors are mostly considered monoclonal neoplasms, the rationale for focused parathyroidectomy in primary hyperparathyroidism. We reported that flow sorting parathyroid tumor cells and methylation-sensitive polymerase chain reaction (me-PCR) of polymorphic human androgen receptor gene and phosphoglycerate kinase gene alleles in deoxyribonucleic acid reveals that ≤35% of parathyroid tumors are polyclonal. We sought to confirm these findings and assess for clinical relevance. Parathyroid tumors from 286 female primary hyperparathyroidism patients were analyzed for clonal status. Tumor clonal status was compared with clinical variables and operative findings. Statistical analysis was performed and significance was established at P < .05. In the study, 176 (62%) patients were informative for human androgen receptor gene and/or phosphoglycerate kinase gene. Assignment of clonal status was made in 119 (68%) tumors, of which 64 (54%) were monoclonal and 55 (46%) were polyclonal. Comparison of tumor clonal status to clinical variables in patients with complete operative data (N = 82) showed that while clinical features were the same between tumor types, patients with polyclonal tumors more often had multiple gland disease (risk ratio 4.066, confidence interval, 1.016-16.26; P = .039) potentially missed at unilateral neck exploration. This work confirms that primary hyperparathyroidism is often the result of polyclonal tumors and that parathyroid tumor clonal status may be associated with multiple gland disease.

1H, 15N, 13C backbone resonance assignments of human phosphoglycerate kinase in a transition state analogue complex with ADP, 3-phosphoglycerate and magnesium trifluoride

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF3−) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO3−), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the 1H, 15N and 13C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF3:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the 1H–15N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.

Virtual Screening against Phosphoglycerate Kinase 1 in Quest of Novel Apoptosis Inhibitors.

Inhibition of apoptosis is a potential therapy to treat human diseases such as neurodegenerative disorders (e.g., Parkinson’s disease), stroke, and sepsis. Due to the lack of druggable targets, it remains a major challenge to discover apoptosis inhibitors. The recent repositioning of a marketed drug (i.e., terazosin) as an anti-apoptotic agent uncovered a novel target (i.e., human phosphoglycerate kinase 1 (hPgk1)). In this study, we developed a virtual screening (VS) pipeline based on the X-ray structure of Pgk1/terazosin complex and applied it to a screening campaign for potential anti-apoptotic agents. The hierarchical filters in the pipeline (i.e., similarity search, a pharmacophore model, a shape-based model, and molecular docking) rendered 13 potential hits from Specs chemical library. By using PC12 cells (exposed to rotenone) as a cell model for bioassay, we first identified that AK-918/42829299, AN-465/41520984, and AT-051/43421517 were able to protect PC12 cells from rotenone-induced cell death. Molecular docking suggested these hit compounds were likely to bind to hPgk1 in a similar mode to terazosin. In summary, we not only present a versatile VS pipeline for potential apoptosis inhibitors discovery, but also provide three novel-scaffold hit compounds that are worthy of further development and biological study.

Lentiviral Vectors Incorporating Ubiquitous Chromatin Opening Element Driving Canine CD18 Expression

Leukocyte adhesion deficiency type 1 (LAD1) in humans is caused due to mutations in the ITGB2 gene encoding the leukocyte CD18 subunit (b2 integrin). This results in defective leukocyte adhesion and migration leading to recurrent episodes of life-threatening bacterial infection. Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific large animal model of LAD1 in which new therapeutic approaches could be tested. Our previous studies have demonstrated variable efficiency of CD18 expression under the control of several promoters. These include cellular promoters such as those of human elongation factor 1a (hEF1a): long (1169bp) and short (248bp) fragments, human phosphoglycerate kinase (hPGK), human CD11b and human CD18 genes. In addition, murine stem cell virus (MSCV) promoter has also been demonstrated to lead to very high levels of CD18 expression in CLAD CD34+ cells thereby reversing the CLAD phenotype in dogs previously treated with both foamy and lentiviral vectors. But, due to potential genotoxicity associated with the use of viral promoters, we continued our efforts in search of novel cellular promoters. One such promoter is the ubiquitous chromatin opening element (UCOE) from the human heterogeneous ribonucleoprotein A2/B1 and chromobox homolog 3 (HNRPA2B1-CBX3) loci. UCOE has been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., Blood 2007).It has also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., Mol Ther. 2010). Since the full-length element is about 2.6 kb, we cloned and tested different fragment lengths of the UCOE promoter in a SIN lentiviral vector (pCL20) in CLAD CD34+ cells in vitro. Efficiency of expression of CD18 obtained with the six promoter fragments of UCOE (in bp), namely U3'631, U3'1262, U3'652, U5'1357, U5'723 and U5'655 were compared to those obtained with an MSCV promoter. Functional viral titers were first determined using a human LAD EBV-transformed B-cell line that lacks endogenous human CD18. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells after a 24h cytokine prestimulation in vitro, the percentage of CD18+ cells 5 days after transduction were as follows: U3'631 - 8.49%, U3'1262 - 15.9%, U3'652 - 21.3% (tested at MOI 100), U5'1357 - 2.05% (tested at MOI 30), U5'723 - 2.44% (tested at MOI 20), U5'655 - 3.01% (tested at MOI 50) and MSCV - 35.3% (tested at MOI 100). The CD18 expression levels driven by some of these promoter fragments were comparable to those driven by cellular promoters mentioned previously. The UCOE is promising in that it could overcome possible gene silencing effects when used in vivo, unlike promoters such as EF1a and PGK which were largely subjected to post-transcriptional gene silencing with sub-therapeutic levels of CD18 as previously tested in the dog model. Hence, functional correction of the CD18 defect could be achieved with candidate UCOE-incorporating SIN lentiviral vector(s) when used in the treatment of CLAD dogs. DisclosuresNo relevant conflicts of interest to declare.

683. Foamy Virus Gene Therapy Significantly Restores Lymphocyte Development and Function in SCID-X1 Mice

X-linked severe-combined immune deficiency (SCID-X1) results from inactivating mutations in the gene encoding the common gamma chain (γc), a cytokine receptor subunit required for lymphoid development and function. In humans, SCID-X1 is characterized by the absence of T cells and natural killer cells, non-functional B cells, and is fatal within the first year of life if left untreated. Although HLA matched allogenic stem cell transplants yield survival rates exceeding 90%, such donors are often unavailable. Thus, gene replacement therapy offers a promising alternative treatment for patients lacking suitable donors. Clinical trials using gamma-retroviral vectors demonstrated efficacy; however, adverse events highlighted the need for improved safety. While adverse events have been eliminated using SIN-gamma-retroviral vectors, we hypothesized that foamy viruses (FV) that lack native pathogenicity and boast an integration site profile much less focused on either active genes or promoter regions than lentiviruses or gamma-retroviruses might provide an improved and safer therapeutic platform. We determined whether a candidate clinical FV vector containing the human phosphoglycerate kinase 1 (PGK) promoter driving human γc expression could safely and effectively rescue lymphocyte development and function in SCID-X1 mice. Our promoter choice was based upon ongoing studies using FV vectors in parallel in a canine model of SCID-X1 where the PGK promoter outperforms the EF1α promoter currently in use in SIN-retro and lentiviral-based SCID-X1 clinical trials. We report here the combined results of 66 primary and 130 secondary transplant mice that demonstrate significant and sustained immune reconstitution. SCID-X1 mice were transplanted with 2×10^6 FV- or LV-transduced, or WT or SCID-X1 lineage negative HSCs. Animals were sacrificed at ~25 weeks post-transplant; bone marrow, spleen and thymus were collected for analysis and secondary recipients were also established. Primary FV gene therapy recipients showed significantly greater B and T lymphoid numbers compared to SCID-X1 controls, with output viral copy numbers of ~1-4 in all tissues. Splenocytes from primary treated animals proliferated in response to CD3/CD28 and also demonstrated γc dependent intracellular pSTAT signaling in response to IL-7 and IL-21. FV and LV gene therapy-treated mice displayed comparable lymphocyte reconstitution and output copy number. Secondary recipients revealed sustained partial rescue of lymphoid compartments and viral marking, indicating the transduction of long-term repopulating HSCs. RIS analysis demonstrated polyclonal marking in splenic lymphoid populations with 3508 unique FV and 2441 unique LV integrations detected (3 experiments/3 animals per experiment). Further, analysis of individual mice from a primary transplant experiment demonstrated polyclonal marking with 588-1060 and 651-895 unique integration sites in FV- and LV-treated animals, respectively. Analysis to determine detailed integration site profile and potential proximity to protooncogenes is ongoing. Together, these data suggest FV gene therapy may provide an effective alternative treatment option for SCID-X1.

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.

X-ray Absorption Spectroscopy and Coherent X-ray Diffraction Imaging for Time-Resolved Investigation of the Biological Complexes: Computer Modelling towards the XFEL Experiment

The development of the next generation synchrotron radiation sources - free electron lasers - is approaching to become an effective tool for the time-resolved experiments aimed to solve actual problems in various fields such as chemistry’ biology’ medicine’ etc. In order to demonstrate’ how these experiments may be performed for the real systems to obtain information at the atomic and macromolecular levels’ we have performed a molecular dynamics computer simulation combined with quantum chemistry calculations for the human phosphoglycerate kinase enzyme with Mg containing substrate. The simulated structures were used to calculate coherent X-ray diffraction patterns’ reflecting the conformational state of the enzyme, and Mg K-edge X-ray absorption spectra, which depend on the local structure of the substrate. These two techniques give complementary information making such an approach highly effective for time-resolved investigation of various biological complexes, such as metalloproteins or enzymes with metal-containing substrate, to obtain information about both metal-containing active site or substrate and the atomic structure of each conformation.

Pgk-Mediated Expression of Common Gamma Chain Is More Effective Than EF1a for Therapeutic Immune Reconstitution of X-SCID Dogs after In Vivo Gene Therapy with Foamy Virus Vector

In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy because they are non-pathogenic in humans, they exhibit increased serum stability and they integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of functional CD3+ lymphocytes in canine X-SCID (Burtner CR et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy that include different promoters and different fluorophores.We performed a head-to-head comparison of two promoters, our previously used EF1α promoter and the human phosphoglycerate kinase (PGK) promoter, by simultaneously injecting three X-SCID pups with equal titers of two therapeutic, human γC-encoding FV vectors. These vectors expressed the fluorophores GFP or mCherry to allow for tracking of transduced cells. Each dog received between 3 and 4 x 108 infectious units of each FV vector. In all treated dogs, lymphocyte marking in the PGK arm reached 50% between day 60 and day 110 post-injection and continued to expand over time, while the EF1α arm peaked at day 42 and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the EF1α arm, suggesting competition within the expanding T cell niche. The development of total CD3+ T cells achieved therapeutic levels (1000 cells/μL of blood) in all three dogs between day 70 and day 130 post-treatment (Fig 1B). We further validated the functionality of these cells by showing that they express a diverse T cell receptor repertoire using spectratyping analysis. In addition, peripheral blood mononuclear cells from the treated animals could be activated in vitro by exposure to the mitogen Phytohemagglutinin A at a level comparable to normal cells. Immunization of the treated dogs with bacteriophage ΦX174 showed production of specific IgG antibodies, suggesting the ability of B lymphocytes to undergo isotype switching. Finally, retroviral integration site analysis revealed polyclonal contribution to the reconstituting T cells. In summary, our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. The outcome in dogs was substantially improved compared to our previous study using EF1α, where robust lymphocyte marking was achieved in only 2 of 5 dogs, and where clonal dominance was observed. Ongoing work will determine whether the superior performance of the PGK vector is due to higher γC expression in PGK vs. EF1α corrected cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus.

BackgroundAdenoviral vector is an efficient tool for gene transfer. Protein expression is regulated by a number of factors, but the regulation by gene copy number remains to be investigated further.ResultsAssessed by flow cytometry, we demonstrated a significant linear correlation between average fluorescence intensity of green fluorescent protein (GFP) and a wide range of multiplicity of infection (MOI), spanning from 0.01 to 200. Average GFP intensity was calculated by mean fluorescence intensity (MFI) × percentage of infection (POI) (MFI × POI) and the correlation was observed in cells transduced with GFP-expressing adenoviral vector driven either by a cytomegalovirus (CMV) promoter for 3 to 6 h or by a human phosphoglycerate kinase (PGK) promoter for 18 to 24 h. Factors impacting this linear correlation include MOI of viral vector, strength of promoter driving GFP expression, cell type transduced and incubation time after gene transfer. We also found that weak GFP signals could be interfered by background signals, whereas strong GFP signals could overshot the detection limitation of the flow cytometer and resulted in a deviation from linearity which was prevented by adjusting the setting in flow cytometer. Moreover, we compared promoter strength as measured by MFI × POI and found that the relative activity of CMV promoter to PGK promoter was 20 to 47 folds in A549 cells and 32 to > 100 folds in H1299 cells.ConclusionsThe linear correlation between MFI × POI and a wide range of adenoviral MOI provides an efficient method to investigate factors regulating protein expression and to estimate virus titers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0137-z) contains supplementary material, which is available to authorized users.

195. Comparative Analysis In Vitro of Regulatory Elements That Drive Targeted Gene Expression in Adenovirus-Associated Viral (AAV) Vectors

Adeno-associated viral (AAV) vectors are a platform with great potential for therapeutic gene delivery. For neurodegenerative disorders such as Friedreich's ataxia, identifying a gene therapy candidate that provides clinically relevant expression of the therapeutic transgene is critical. Friedreich's ataxia is caused by the decreased expression of the mitochondria matrix protein frataxin (FXN) due to mutations in the frataxin gene. Elevation of FXN protein in the nervous system is an approach that is likely to be beneficial in addressing the neurological components of this devastating disease. However, the properties of regulatory elements that drive exogenous gene expression from AAV vectors have not been well characterized. In this study, a set of AAV vectors were generated with various combinations of different regulatory elements directing the expression of the human frataxin gene. These regulatory elements included the canonical Kozak sequence, mitochondria targeting sequences, simian virus 40 (SV40) intron, AAV inverted internal repeat (ITR), and constitutive promoters such as cytomegalovirus (CMV), chicken b-actin (CBA), human frataxin, human phosphoglycerate kinase-1 (PGK), human synapsin and human elongation factor 1α (EF-1α) promoters. For comparison in vitro, these AAV constructs were introduced into three types of human cells and primary rat DRG neurons by co-transfection with a GFP expression construct which was used as an internal control. FXN and GFP protein expression were quantitatively measured by ELISA and the relative level of FXN expression calculated by normalizing to the level of GFP in each condition. These studies demonstrated that the CMV promoter is the most potent in driving FXN expression followed by the CBA promoter. Much lower FXN expression levels were observed when the endogenous frataxin or PGK promoter was used. The canonical Kozak sequence, the SV40 intron alone and different mitochondria targeting sequences had no significant impact on FXN expression. In addition, we evaluated the effect of different optimized human codons within the frataxin transgene on FXN expression level in the context of the CBA promoter and SV40 intron. Our results demonstrated that several of these codon-optimized frataxin genes consistently increased the expression of FXN protein at least 2-fold compared to the wild type frataxin gene. Together, these results provide important information for guiding the selection and optimization of regulatory elements and codons for an AAV gene therapy for Friedreich's ataxia.