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Objective It has been reported that crocodile blood contains potent antibacterial and antiviral properties. However, its effects on HIV-1 infection remain unknown. Design We obtained blood from saltwater crocodiles to examine whether serum or plasma could inhibit HIV-1 infection. We purified plasma fractions then used liquid chromatography-mass spectrometry to identify the inhibitory protein factor(s). We then analyzed the ability of recombinant proteins to recapitulate HIV-1 inhibition and determine their mechanism of action. Methods Crocodylus porosus plasma was tested for inhibition of Jurkat T-cell HIV-1 infection. Inhibitor(s) were purified by reverse-phase chromatography then identified by protein liquid chromatography-mass spectrometry. Anti-HIV-1 activity of purified plasma or recombinant proteins were measured by p24 enzyme-linked immunosorbent assay and luciferase readouts, and mechanism of action was determined by measuring HIV-1 RNA, cDNA and transcription (using 1G5 cells). Results Crocodile plasma contains potent inhibitors of HIV-1IIIB infection, which were identified as histones. Recombinant human histones H1 and H2A significantly reduced HIV-1JR-FL infection (IC50 of 0.79 and 0.45 μmol/l, respectively), whereas H4 enhanced JR-FL luciferase activity. The inhibitory effects of crocodile plasma, recombinant H1 or recombinant H2A on HIV-1 infection were during or post-viral transcription. Conclusion Circulating histones in crocodile blood, possibly released by neutrophil extracellular traps, are significant inhibitors of HIV-1 infection in-vitro. Extracellular recombinant histones have different effects on HIV-1 transcription and protein expression and are downregulated in HIV-1 patients. Circulating histones may be a novel resistance factor during HIV-1 infection, and peptide versions should be explored as future HIV-1 therapeutics that modulate viral transcription.

Summary Previously, we have reported that interleukin (IL)-4, granulocyte–macrophage colony-stimulating factor (GM-CSF), and IL-11, but not IL-33, are up-regulated in two strains of mice with immune thrombocytopenia (ITP) that are responsive to intravenous immunoglobulin (IVIg) treatment. Previously, IL-4 was ruled out in the mechanism of IVIg; however, other publications have suggested this cytokine as a major player in the mechanism of IVIg action. Thus, we sought to further investigate a role for IL-4 and, in addition, GM-CSF and IL-11 in the mechanism of action of IVIg using a murine model of ITP. A passive platelet antibody model was used to generate ITP in IL-4 receptor knock-out (IL-4R–/–), IL-11 receptor knock-out (IL-11Rα–/–) and GM-CSF knock-out (Csf2–/–) mice. We also used a neutralizing antibody to IL-11 and recombinant human IL-11 (rhIL-11) in addition to depleting basophils in vivo to study the effect of IVIg to ameliorate ITP. Our results showed that basophils, IL-4 and GM-CSF were unimportant in both ITP induction and its amelioration by IVIg. The role of IL-11 in these processes was less clear. Even though IL-11Rα–/– mice with ITP responded to IVIg similarly to wild-type (WT) mice, treatment of ITP WT mice with rhIL-11 instead of IVIg showed an increase in platelet numbers and WT mice administered anti-IL-11 showed a significant reduction in the ability of IVIg to ameliorate the ITP. Our findings indicate that neither IL-4, basophils or GM-CSF have roles in IVIg amelioration of ITP; however, a role for IL-11 requires further study.

WOS: 000396610300030, We prove that some integrals of Bessel functions are transcendence over ring of Bessel functions with coefficients from the field of rational fractions of one variable. (C)2017 All rights reserved.

Targeting the androgen signalling pathway has long been the hallmark of anti-hormonal therapy for prostate cancer. However, development of androgen-independent prostate cancer is an inevitable outcome to therapies targeting this pathway, in part, owing to the shift from cancer dependence on androgen signalling for growth in favor of augmentation of other cellular pathways that provide proliferation-, survival- and angiogenesis-promoting signals. This review focuses on the role of the hormone relaxin in the development and progression of prostate cancer, prior to and after the onset of androgen independence, as well as its role in cancers of other reproductive tissues. As the body of literature expands, examining relaxin expression in cancerous tissues and its role in a growing number of in vitro and in vivo cancer models, our understanding of the important involvement of this hormone in cancer biology is becoming clearer. Specifically, the pleiotropic functions of relaxin affecting cell growth, angiogenesis, blood flow, cell migration and extracellular matrix remodeling are examined in the context of cancer progression. The interactions and intercepts of the intracellular signalling pathways of relaxin with the androgen pathway are explored in the context of progression of castration-resistant and androgen-independent prostate cancers. We provide an overview of current anti-hormonal therapeutic treatment options for prostate cancer and delve into therapeutic approaches and development of agents aimed at specifically antagonizing relaxin signalling to curb tumor growth. We also discuss the rationale and challenges utilizing such agents as novel anti-hormonals in the clinic, and their potential to supplement current therapeutic modalities.

Androgen hormones and the androgen receptor (AR) pathway are the main targets of anti-hormonal therapies for prostate cancer. However, resistance inevitably develops to treatments aimed at the AR pathway resulting in androgen-independent or hormone-refractory prostate cancer (HRPC). Therefore, there is a significant unmet need for new, non-androgen anti-hormonal strategies for the management of prostate cancer. We demonstrate that a relaxin hormone receptor antagonist, AT-001, an analog of human H2 relaxin, represents a first-in-class anti-hormonal candidate treatment designed to significantly curtail the growth of androgen-independent human prostate tumor xenografts. Chemically synthesized AT-001, administered subcutaneously, suppressed PC3 xenograft growth by up to 60%. AT-001 also synergized with docetaxel, standard first-line chemotherapy for HRPC, to suppress tumor growth by more than 98% in PC3 xenografts via a mechanism involving the downregulation of hypoxia-inducible factor 1 alpha and the hypoxia-induced response. Our data support developing AT-001 for clinical use as an anti-relaxin hormonal therapy for advanced prostate cancer.

Objective: We investigated whether HIV-1 inhibition by SRC-family kinase inhibitors is through the non-receptor tyrosine kinase pp60c-SRC (c-SRC) and its binding partner, protein tyrosine kinase 2 beta (PTK2B). Design: CD4+ T-lymphocytes were infected with R5 (JR-FL) or X4 (HXB2) HIV-1. We used SRC-family kinase inhibitors or targeted siRNA knockdown of c-SRC and PTK2B, then monitored effects on the early HIV-1 lifecycle. Methods: Four SRC-family kinase inhibitors or targeted siRNA knockdown were used to reduce c-SRC or PTK2B protein expression. Activated CD4+ T-lymphocytes were infected with recombinant, nef-deficient, or replication-competent infectious viruses. Knockdown experiments examined early infection by monitoring: luciferase activity, expression of host surface receptors, reverse transcriptase activity, p24 levels and qPCR of reverse transcripts, integrated HIV-1, and two-long terminal repeat (2-LTR) circles. Results: All SRC-family kinase inhibitors inhibited R5 and X4 HIV-1 infection. Neither c-SRC nor PTK2B siRNA knockdown had an effect on cell surface receptors (CD4, CXCR4, and CCR5) nor on reverse transcriptase activity. However, using JR-FL both decreased luciferase activity while increasing late reverse transcripts (16-fold) and 2-LTR circles (eight-fold) while also decreasing viral integration (four-fold). With HXB2, c-SRC but not PTK2B siRNA knockdown produced similar results. Conclusions: Our results suggest c-SRC tyrosine kinase is a major regulator of HIV-1 infection, participating in multiple stages of infection post-entry: Reduced proviral integration with increased 2-LTR circles is reminiscent of integrase inhibitors used in combination antiretroviral therapy. Decreasing c-SRC expression and/or activity provides a new target for antiviral intervention and the potential for repurposing existing FDA-approved kinase inhibitors.

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies. Fil: Scaife, Matthew. University of Toronto; Canadá Fil: Pacienza, Natalia Alejandra. University Health Network. Ontario Cancer Institute; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Au, B. C. Y.. University Health Network. Ontario Cancer Institute; Canadá Fil: Wang, J. C. M.. University Health Network. Ontario Cancer Institute; Canadá Fil: Devine, S.. University of Toronto; Canadá Fil: Scheid, E.. Mc Master University; Canadá Fil: Lee, C. J.. University Health Network. Ontario Cancer Institute; Canadá Fil: Lopez Perez, O.. University Health Network. Ontario Cancer Institute; Canadá Fil: Neschadim, A.. University of Toronto; Canadá Fil: Fowler, D. H.. National Institutes of Health; Estados Unidos Fil: Foley, R.. Mc Master University; Canadá Fil: Medin, J. A.. University of Toronto; Canadá. University Health Network. Ontario Cancer Institute; Canadá

Hematopoietic stem cells comprise a prominent target for gene therapy aimed at treating various genetic and acquired disorders. A number of limitations associated with hematopoietic cell transplantation can be circumvented by the use of cells stably modified by retroviral gene transfer. Oncoretroviral and lentiviral vectors offer means for generating efficient and stable transgene expression. This review summarizes the state of the field today in terms of vector development and clinical experimentation. In particular, concerns with the safety of retroviral vectors intended for clinical gene transfer, applicability of preclinical data in directing clinical trial design, and recent research aimed at resolving some of these issues are addressed. Finally, this review underlines the specific advantages offered by lentiviral gene-transfer vectors for gene therapy in stem cells.

Gene therapy and stem cell transplantation safety could be enhanced by control over the fate of therapeutic cells. Suicide gene therapy uses enzymes that convert prodrugs to cytotoxic entities; however, heterologous moieties with poor kinetics are employed. We describe a novel enzyme/prodrug combination for selectively inducing apoptosis in lentiviral vector-transduced cells. Rationally designed variants of human thymidylate kinase (tmpk) that effectively phosphorylate 3'-azido-3'-deoxythymidine (AZT) were efficiently delivered. Transduced Jurkat cell lines were eliminated by AZT. We demonstrate that this schema targeted both dividing and non-dividing cells, with a novel killing mechanism involving apoptosis induction via disruption of the mitochondrial inner membrane potential and activation of caspase-3. Primary murine and human T cells were also transduced and responded to AZT. Furthermore, low-dose AZT administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced K562 cells suppressed tumor growth. This novel suicide gene therapy approach can thus be integrated as a safety switch into therapeutic vectors.

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN–expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1–2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab′)2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

Експериментально досліджено процеси згрудкування і випалення залізорудних котунів з різним розподілом твердого палива в пошаровій системі. З’ясовано вплив функціонального розподілення твердого палива на міцність при стисканні і ступінь металізації випалених котунів. Міцність на стискання випалених котунів основністю 1,4, що містять підвищену кількість дисперсного палива, змінюється в межах 1,6—2,35 кН/котун і не характеризується високими показниками, хоча і перевищує вимоги держстандарту на 0,2—0,95 кН/котун. Перерозподіл твердого палива характеризується тим, що переважна кількість його мітиться всередині гранул (12 %)і лише 3,3 % — у міжгранульному просторі. В результаті цього істотно підвищується ступінь металізації спеченого матеріалу, зменшується теплове навантаження на реакційний вузол електропечі опору і дещо знижується міцність на стискання випалених котунів. Максимальний ріст ступеня металізації (38,2 %) спостерігається за наявності на їх поверхні захисного шару із суміші негашеного вапна і залізорудного концентрату. Захисна оболонка слугує додатковим бар’єром, який у процесі спікання й охолодження котунів перешкоджає вільному доступу кисню до вуглецю, що міститься всередині гранул, значно зменшуючи інтенсивність реакції окиснення твердого палива. В результаті підвищується ступінь металізації шихтового матеріалу, зменшується витікання розплаву із гранул, що не призводить до сплавлення їх у вигляді шматків великого розміру. Підвищення ступеня металізації випалених котунів супроводжується збільшенням концентрації металевого заліза у вигляді “глобульних частинок” усередині гранул і монооксиду заліза у поверхневому шарі. In this article processes of pelletizing and sintering iron ore pellets with different distribution of solid fuel in multilayer system were experimentally investigated. The influence of the functional distribution of solid fuels on the compressive strength and the degree of metallization fired pellets was studied. Compressive strength of burnt pellets with basicity of 1.4, which contain increased amounts of particulate fuel in range of 1,6—2,35 kN/pellet and not characterized by high indices, although GOST has indices on level 0,2—0,95 kN/pellet. Reallocation of a solid fuel is characterized by concentration of fuel in granules themselves (12 %) and only 3,3 % in space between the pellets. As a result, this significantly increases the degree of metallization of the calcined material, reducing the thermal load on the electric unit of the reaction site of electric furnace and slightly reduces compressive strength of burnt pellets. Maximum increase in the degree of metallization (38,2 %) of burnt pellets is observed in the presence of the protective layer of a quick lime and iron ore mixture on the surface. The protective shell serves as an additional barrier which, during passing the firing and cooling the pellets, prevents the free access of oxygen to the carbon particles located within the granules, substantially reducing the intensity of the oxidation reaction of the solid fuel. As result this increases the degree of metallization of the charge material and decreases outflow of melt from the pellets burning in this moment, and it does not lead to fusing them into large lumps. Increasing degree of metallization of burnt pellets is accompanied by an increase in the concentration of metallic iron in the form of “globular particles” in the nucleus, and granules of iron monoxide in the surface layer. Экспериментально исследованы процессы окомкования и обжига железорудных окатышей с различным распределением твердого топлива в послойной системе. Изучено влияние функционального распределения твердого топлива на прочность при сжатии и степень металлизации обожженных окатышей. Прочность на сжатие обожженных окатышей основностью 1,4, которые содержат повышенное количество дисперсного топлива, изменяется в пределах 1,6—2,35 кН/окатыш и не характеризуется высокими показателями, хотя и превышает требования гостстандарта на 0,2—0,95 кН/окатыш. Перераспределение твердого дисперсного топлива характеризуется тем, что большая часть топлива сосредоточена в самих гранулах (12 %) и лишь 3,3 % — в межгранульном пространстве. В результате этого существенно повышается степень металлизации обожженного материала, уменьшается тепловая нагрузка на реакционный узел электропечи сопротивления и несколько понижается прочность на сжатие обожженных окатышей. Максимальный рост степени металлизации (38,2 %) обожженных окатышей наблюдается при наличии на их поверхности защитного слоя из смеси негашеной извести и железорудного концентрата. Защитная оболочка служит дополнительным барьером, который в процессе обжига и охлаждения окатышей препятствует свободному доступу кислорода к углеродным частицам, которые расположены внутри гранул, существенно уменьшая интенсивность реакции окисления твердого топлива. В результате повышается степень металлизации шихтового материала, уменьшается вытекание расплава из гранул, что не приводит к сплавлению их в виде кусков большого размера. Повышение степени металлизации обожженных окатышей сопровождается увеличением концентрации металлического железа в виде “глобульных частиц” в ядре гранул и монооксида железа в поверхностном слое.

We previously described a novel suicide (or ‘cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

Експериментально досліджено чотири способи отримання котунів з рудно-флюсових композицій з підвищеним вмістом твердого палива, що базуються на поєднанні процесів згрудкування вихідних сировинних матеріалів і їх спікання за температури 1593-1598 К: 1) тверде паливо змішувалося з вихідними шихтовими матеріалами безпосередньо перед їх згрудкуванням; 2) паливо накочувалось на поверхню гранул крупністю 10-15 мм, і лише потім котуни підлягали випаленню; 3) варіантні частинки твердого палива (50 %) змішувались із сировинними шихтовими матеріалами перед їх згрудкуванням, а решта накочувалась на поверхню гранул з подальшою імітацією реального процесу змішування котунів із твердим паливом у барабанному змішувачі; 4) тверде паливо в кількості 40 % змішувалось із сировинними шихтовими матеріалами перед їх згрудкуванням, 40 % твердого палива наносилось на поверхню сирих гранул з імітацією процесу барабанного згрудкування, а 20 % залишкового палива наносилось на гранули перед завантаженням їх у піч. Міцність на стиснення випалених офлюсо-ваних котунів, що містять підвищену кількість твердого порохнявого палива, змінюється в межах 1,9-2,45 кН/котун і не характеризується високими показниками, хоча і перевищує вимоги Держстандарту на 0,5-0,95 кН/котун. Визначено вміст монооксиду заліза (FeO) і заліза металевого (Feмет) в офлюсованих котунах після їх випалення і встановлено, що FeO змінюється в межах 21,5-27,2 %, Feмет - 11,2-18,9 %. Максимальний ріст цих показників спостерігається у випалених котунах, виготовлених за четвертим варіантом. Підвищення вмісту FeO і особливо Feмет у компактному випаленому шихтовому матеріалі свідчить, що застосування такої технології сприяє росту ступенів дисоціації гематиту і металізації в кінцевому продукті та підвищенню його металургійних властивостей. Four methods of obtaining pellets with fluxing ore compositions with higher content of solid fuel, based on the combination of feedstock sintering process and sintering at a temperature of 1593-1598 K, namely: 1)a method wherein the solid fuel was mixed with the initial charge materials directly before agglomeration; 2) a method wherein the solid fuel was rolled on the surface of granules of size 10-15 mm, and after that pellets were burned out; 3) the method in which varying particles solid fuel (50 %) were also mixed with the raw materials before sintering, while the second part of them was rolled on the surface of the granules, followed by imitation of the pellets real process of mixing with solid fuel in a drum mixer; 4) the method in which a solid fuel in an amount of 40 % was mixed with raw materials before sintering were experimentally investigated. Compression strength of burned out fluxing pellets with the higher content of powdered solid fuel, is changed within 1,9-2,45 kN/pellet and is not characterized as high index though it exceeds the requirements of GOST on 0,5-0,95 kN/pellet. The content of iron monoxide (FeO) and iron metal (Femet) in fluxed pellets after burning out was found and it was established, that FeO varies in range of 21,5-27,2 %, whereas Femet varies in range of 11,2-18,9 %. The maximum increase of these indexes was observed in the burned out pellets made according to the fourth method. Increasing the content of iron monoxide and, particularly, iron in the compact metallic charge materials indicates that the use of such technology is accompanied by the increasing of the hematite dissociation degree and plating in the final product and improving its metallurgical properties. Экспериментально исследованы четыре способа получения окатышей с рудно-флюсовых композиций с повышенным содержанием твердого топлива, основанные на сочетании процессов окускования исходных сырьевых материалов и их спекания при температуре 1593-1598 К: 1) твердое топливо смешивалось с исходными шихтовыми материалами непосредственно перед их окускованием; 2) твердое топливо накатывалось на поверхность гранул крупностью 10-15 мм, и только потом окатыши подлежали обжигу; 3) вариантные частицы твердого топлива (50 %) смешивались с сырьевыми шихтовыми материалами перед их окускованием, а остальная часть накатывалась на поверхность гранул с последующей имитацией реального процесса смешивания окатышей с твердым топливом в барабанном смесителе; 4) твердое топливо количестве 40 % смешивалось с сырьевыми шихтовыми материалами перед их окускованием, 40 % твердого топлива наносилось на поверхность сырых гранул с имитацией процесса барабанного окускования, а 20 % остаточного топлива наносилось на гранулы перед загрузкой их в печь. Прочность на сжатие обожженных офлюсованных окатышей, содержащих повышенное количество твердого пылевидного топлива, изменяется в пределах 1,9-2,45 кН/окатыш и не характеризуется высокими показателями, хотя превышает требования ГОСТа на 0,5-0,95 кН/окатыш. Определено содержание монооксида железа (FeO) и железа металлического (Feмет) в офлюсованных окатышах после их обжига и установлено, что FeO изменяется в пределах 21,5-27,2 %, Feмет - 11,2-18,9 %. Максимальный рост данных показателей наблюдается в обожженных окатышах, изготовленных по четвертому варианту. Повышение содержания FeO и особенно Feмет в компактном обожженном шихтовом материале свидетельствуют о том, что применение такой технологии сопутствует росту степеней диссоциации гематита и металлизации в конечном продукте и повышению его металлургических свойств.

Activity and specificity of chemotherapeutic agents against solid tumors can be augmented via the targeted or localized delivery of ‘suicide’ genes. Selective activation of specific prodrugs in cells expressing the ‘suicide’ gene drives their elimination by apoptosis, while also enabling the killing of adjacent bystander cells. Strong bystander effects can compensate for poor ‘suicide’ gene delivery, and depend on the prodrugs used and mechanisms for the acquisition of activated drug by the bystander population, such as the presence of gap junctional intercellular communications. Although a number of ‘suicide’ gene therapies for cancer have been developed and characterized, such as herpes simplex virus-derived thymidine kinase (HSV-tk)-based activation of ganciclovir, their limited success highlights the need for the development of more robust approaches. Limiting activation kinetics and evolution of chemoresistance are major obstacles. Here we describe ‘suicide’ gene therapy of cancer based on the lentivirus-mediated delivery of a thymidine-active human deoxycytidine kinase variant. This enzyme possesses substrate plasticity that enables it to activate a multitude of prodrugs, some with distinct mechanisms of action. We evaluated the magnitude and mechanisms of bystander effects induced by different prodrugs, and show that when used in combination, they can synergistically enhance the bystander effect while avoiding off-target toxicity.

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

Farber disease is a rare lysosomal storage disorder (LSD) that manifests due to acid ceramidase (AC) deficiencies and ceramide accumulation. We present a preclinical gene therapy study for Farber disease employing a lentiviral vector (LV-huAC/huCD25) in three enzymatically normal nonhuman primates. Autologous, mobilized peripheral blood (PB) cells were transduced and infused into fully myelo-ablated recipients with tracking for at least 1 year. Outcomes were assessed by measuring the AC specific activity, ceramide levels, vector persistence/integration, and safety parameters. We observed no hematological, biochemical, radiological, or pathological abnormalities. Hematological recovery occurred by approximately 3 weeks. Vector persistence was observed in PB and bone marrow (BM) cells by qualitative and quantitative PCR. We did not observe any clonal proliferation of PB and BM cells. Importantly, AC-specific activity was detected above normal levels in PB and BM cells analyzed post-transplantation and in spleens and livers at the endpoint of the study. Decreases of ceramide in PB cells as well as in spleen and liver tissues were seen. We expect that this study will provide a roadmap for implementation of clinical gene therapy protocols targeting hematopoietic cells for Farber disease and other LSDs.

In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET.Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs.Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine).The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

Donor Lymphocyte Infusion (DLI) is employed in the treatment of various malignancies, as donor-derived allogeneic T lymphocytes elicit strong anti-tumor immune responses (GvL/GvT). Unfortunately, these are often accompanied by GvHD, mediated by donor T cells that are allo-activated against host tissues. GvHD manifests with immunosupression, multi-organ dysfunction, severe morbidity and high mortality. It is suggested that CD4+Th1 and CD8+Tc1 cell subsets mediate both strong GvL/GvT and strong GvHD, while being cross-regulated by CD4+Th2 and CD8+Tc2 cells that mediate only moderate effects. Patients transplanted with T cell-depleted BM experience little GvHD, but have high rates of cancer relapse. It is possible to maintain beneficial GvL/GvT responses while controlling GvHD by transferring a drug-activating ‘suicide’ gene into donor T cells prior to transplant. Cells expressing the ‘suicide’ gene-product convert a non-toxic prodrug into a cytotoxic antimetabolite, and are eliminated by apoptosis. We have constructed 3rd generation lentiviral vectors (LVs) for the expression of human converting enzymes. We are using novel enzymes, endogenously present in human cells, to avoid unwanted immune responses against vector-transduced cells, recently proven a major drawback in clinical trials. Our approaches are based on the human thymidylate kinase (Tmpk), which acts on Zidovudine (AZT), and the human deoxycytidine kinase (dCK), which potently activates a number of prodrugs, including Cytarabine (AraC), Gemcitabine and Cladribine. We have developed active-site engineered mutants of Tmpk and dCK that are up to two orders of magnitude more catalytically active than wild-type (wt) enzymes. Our LVs efficiently transduce both immortalized and primary cells (up to 99%). The LV expression cassette also encodes a non-signaling form of human CD19 molecule that serves as a marker for ex vivo enrichment and in vivo tracking of transduced cells. We demonstrate efficient, selective and prompt killing of transduced cells in a dose-dependent manner by a number of prodrugs in cell lines in vitro and in a murine solid tumor model in vivo. For example, R16GLL Tmpk mutant-transduced Jurkat cells (human T cell leukemia line) are completely killed by apoptosis within 4 days of culture in media containing 100uM AZT (IC50 of 2μM), while wt cells are unaffected at the same conditions. In a NOD/SCID murine tumor model, K562 erythroid leukemia cells transduced with either empty vector, wt Tmpk, and R16GLL or F105Y Tmpk mutants were implanted subcutaneously. Mice bearing tumor cells expressing either R16GLL or F105Y Tmpk and receiving a low daily dose of 2.5mg/kg of AZT have developed significantly reduced tumors following 2 weeks of treatment (tumor size of 24–200mm2), compared to mice bearing wt Tmpk and empty vector transduced cells, or untreated mice (tumor size of over 1000mm2). We are currently evaluating the efficiency with which transduced cells can be cleared from circulation in a murine leukemia model and are evaluating the complete GvHD-treatment strategy in a murine model of GvHD/GvT based on the CD45 congenic strains. This much improved therapeutic strategy may offer a safe and complete solution for GvHD in patients undergoing BMT.

Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide’ strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.

The infusion of donor lymphocytes in allogenic BMT recipients provides potent antitumor activity to treat recurrent malignancies. One complication, however, is severe GvHD, which is mediated by T cells in the graft. One approach to control GvHD is to employ ‘suicide’ gene therapy. In the customary adaptation of this approach, the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene, combined with the antiviral prodrug ganciclovir (GCV), is used to control GvHD after introduction of this suicide gene into donor T lymphocytes. However, the efficiency of HSV1-tk is suboptimal and the issue of host immunogenicity against this heterologous effector gene product can hamper outcomes. In addition, prophylactic GCV is often used to control cytomegalovirus infection after BMT. This confounds the broad clinical implementation of this approach. Our novel suicide gene therapy strategy combines the use of human thymidylate monophosphate kinase (tmpk) and the prodrug Zidovudine (AZT) in a lentiviral vector (LV) format. Since tmpk is endogenously expressed in human cells, immunogenic responses will be limited. We constructed a LV expression system carrying wild-type or one of two modified forms of tmpk. These engineered tmpk mutants (F105Y and BL) show substantially increased catalytic conversion of AZT compared to wild-type tmpk. Our vector also includes a truncated form of human CD19 (hCD19Δ), not normally expressed on the T cell lineage, that can be used to enrich and track transduced cells. Highly efficient (95%) transduction of Jurkat cells (human T cell leukemia line) was attained by a single infection with our LVs (MOI of 10). Transduced cells were efficiently and selectively killed in a dose-dependent manner by AZT (IC50 of 2 μM), while wild-type tmpk transduced cells were unaffected by AZT up to 100 μM. In response to AZT treatment, the apoptotic cell indices of cells transduced with wild-type tmpk, F105Y, or BL were 6.2 ± 0.3 %, 40.7 ± 1.7 %, and 46.1 ± 4.6 %, respectively (n=3). We next established by HPLC that cells transduced with a LV encoding a mutant form of tmpk effectively convert AZT into its active anti-metabolite form, AZT-triphosphate (AZT-TP). Intracellular ratio of AZT-TP to AZT-monophosphate (MP) is 11.3 in cells transduced with a LV encoding the BL mutant of tmpk, compared to 0.02 in non-transduced cells and 0.10 in wild-type tmpk transduced cells. Our findings also revealed that following incubation with indirubin-3′-oxime, which inhibits cellular proliferation, and AZT treatment, transduced cells were successfully killed. Thus the cytotoxic mechanism differs from HSV1-tk mediated cell killing and is independent of cell proliferation. We also succeeded in the infection of primary mouse and human T cells to over 40% and 70% transduction efficiency, respectively. Lastly, we have shown that in vivo growth of tumor cells transduced with these mutant tmpk LVs was totally inhibited by treatment with AZT. These results demonstrate that our novel suicide gene therapy system has significant potential for many clinical applications.