Pharmacokinetic Analysis of Omomyc Shows Lasting Structural Integrity and Long Terminal Half-Life in Tumor Tissue

Omomyc; Mass spectrometry; Protein therapeuticsOmomyc; Espectrometría de masas; Terapéutica de proteínasOmomyc; Espectrometria de masses; Terapèutica de proteïnesMYC is an oncoprotein causally involved in the majority of human cancers and a most wanted target for cancer treatment. Omomyc is the best-characterized MYC dominant negative to date. In the last years, it has been developed into a therapeutic miniprotein for solid tumor treatment and recently reached clinical stage. However, since the in vivo stability of therapeutic proteins, especially within the tumor vicinity, can be affected by proteolytic degradation, the perception of Omomyc as a valid therapeutic agent has been often questioned. In this study, we used a mass spectrometry approach to evaluate the stability of Omomyc in tumor biopsies from murine xenografts following its intravenous administration. Our data strongly support that the integrity of the functional domains of Omomyc (DNA binding and dimerization region) remains preserved in the tumor tissue for at least 72 hours following administration and that the protein shows superior pharmacokinetics in the tumor compartment compared with blood serum.This research has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 872212 and from the Ministerio de Ciencia e Innovacion under grant no. RTC2019-007067-1

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics

Nanovesicles; Neuroblastoma; Pediatric cancerNanovesículas; Neuroblastoma; Cáncer pediátricoNanovesícules; Neuroblastoma; Càncer pediàtricMicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.The funding was received by Ministerio de Educación, Cultura y Deporte (Grant no. FPU16/01099), Ministerio de Economía, Industria y Competividad (Grants MAT2016-80820-R, MAT2016-80826-R and SAF2016-75241-R), the Ministry of Science and Innovation (MINECO) of Spain through grant PID2019-105622RB-I00, from Instituto de Salud Carlos III (Grant no. CP16/00006, PI17/00564, PI20/00530, DTS20/00018) (Co-funded by European Regional Development Fund/European Social Fund) “Investing in your future”), from the EuroNanoMed II platform through the NanoVax project, from CIBER-BBN through grant TAG-SMARTLY, Joan Petit Foundation, Asociación Matem Lo Bitxo and Asociación Española Contra el Cáncer (Grant no. LABAE18009SEGU), as well as, Generalitat de Catalunya through the Centres de Recerca de Catalunya (CERCA) programme and grant no. 2017-SGR-918, and from Agency for Management of University and Research Grants (AGAUR) (Grant no 2018LLAV0064 and SIFECAT IU68-010017). Furthermore, ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S)

Anàlisi de l’expressió gènica en els ganglis de les arrels dorsals del nervi ciàtic en el ratolí transgènic rip/infβ, model de neuropatia diabètica

La polineuropatia diabètica (PND) és una de les complicacions secundàries més freqüents de la diabetis mellitus. Provoca un empobriment de la qualitat de vida dels malalts, causa dolor i úlceres i, en última instància, amputació de l’extremitat afectada. La patogènia és multifactorial, hi ha alteracions estructurals en els nervis perifèrics i els tractaments actuals són simptomàtics, no específics i poc efectius. El ratolí transgènic RIP/INFβ és un model de diabetis, que mimetitza la diabetis autoimmune humana, provocant la destrucció de les cèl·lules β pancreàtiques, que s’intensifica amb l’administració de dosis baixes repetides d’estreptozotocina (STZ). Partint de la base de que els processos biològics implicats en la degeneració i regeneració dels nervis perifèrics estan controlats pels ganglis de les arrels dorsals (GAD), l’objectiu d’aquesta tesi ha estat analitzar l’expressió gènica i els processos biològics que esdevenen en els GAD del nervi ciàtic després d’un traumatisme, en el ratolí transgènic RIP/INFβ tractat amb dosis baixes i repetides de STZ (Tg-STZ). S’ha treballat amb els GAD del nervi ciàtic de ratolins, en diferents condicions experimentals: controls ICR (ICR) atès que els ratolins transgènics presenten aquest fons genètic; ICR tractats amb STZ (ICR-STZ); transgènics RIP/INFβ (Tg); i Tg tractats amb STZ (Tg-STZ). Quatre setmanes després de considerar-ne instaurada la diabetis en els Tg-STZ, tots els animals van ser sotmesos a una lesió per aixafament en el nervi ciàtic de l’extremitat esquerra, mantenint la dreta intacta. Quatre setmanes desprès de la lesió es van prendre mostres dels GAD del nervi ciàtic d’ambdues extremitat. Es va extreure el RNA dels GAD per realitzar els estudis de microarrays (Affymetrix GeneChip® Mouse Genome 430 2.0 Array). Per validar els valors de l’expressió gènica es va realitzar una PCR quantitativa en temps real (RT-qPCR). Es van realitzar comparacions dels perfils gènics entre les diferents condicions experimentals: 1) ICR-STZ lesionades vs ICR lesionades; 2) ICR lesionades vs ICR no lesionades; 3) Tg-STZ no lesionades vs ICR no lesionades; i 4) Tg-STZ lesionades vs ICR lesionades. Les mostres de referència van ser els GAD de l’extremitat no lesionada dels ratolins ICR (baseline) amb un fold change (FC) de 1. Un cop obtinguts els perfils de les comparatives es van analitzar els gens sobrerregulats i infrarregulats, i els processos biològics implicats, amb diferents bases de dades (Database for Annotation, Visulaization and Integrated Discovery; Center for Quantitaive Biology; Gene Home). L’absència de diferències en l’expressió gènica entre els GAD de les extremitats lesionades dels animals ICR-STZ vs ICR, indica que, en aquests òrgans, la STZ no té un efecte neurotòxic. En animals control ICR a les 4 setmanes després d’una lesió en el nervi ciàtic, encara es sobrexpressen gens relacionats amb la regeneració nerviosa, que participen en les vies de senyalització (Gpr151, Nts), transmissió sinàptica (Npy), projeccions neuronals i guia de l’axó (Sox11, Atf3, Tnc, Sema6a, Sprr1a, Gal), el que indicaria que la lesió encara no està totalment resolta. En animals transgènics (Tg-STZ) diabètics, la infrarregulació de gens relacionats amb vies metabòliques (Vgf) i la sobrerregulació de gens implicats en el metabolisme de hidrats de carboni (Car3) i lípids (Gdpd3), indicaria que a les 8 setmanes d’iniciar el procés diabètic els GAD estarien afectats per la hiperglucèmia. En animals transgènics (Tg-STZ) diabètics, 8 setmanes després de començar la hiperglucèmia, la infrarregulació de gens implicats en desenvolupament del sistema nerviós (Gal, Bdnf, Erg3), regeneració axonal (Sprr1a, Lingo1), transmissió sinàptica (Calb1, Snapin), transducció de senyals (Nts, Rgs2), canals de potassi (Kcns1, Kcnq5, Knj13, Kcna6, Kcnt1) i apoptosi (Tfnaip, Ctsb, E2f1, Crh), indicaria l’existència de canvis degeneratius en els GAD deguts a la neuropatia diabètica. En animals transgènics (Tg-STZ) diabètics, 4 setmanes després d’una lesió per aixafament del nervi ciàtic, la sobreexpressió de gens relacionats amb guia de l’axó (Foxd1), regeneració axonal (Sprr1a), projeccions neuronals (Mapk8), supervivència de les neurones dels GAD i elongació de les neurites (Gal, Sox11), o de vies de senyalització nerviosa (Npy1r, Igf1, Rgs18, Gpr151), transmissió sinàptica (Gabrb3, Neto1, CcKbr) i apoptosi (Crh), assenyalaria que en els GAD hi ha processos de regeneració posttraumàtica. En animals transgènics (Tg-STZ) diabètics, 4 setmanes després d’una lesió per aixafament del nervi ciàtic, la infraexpressió de gens relacionats amb l’axogènesi (Nptx1, Cxcr4), transmissió sinàptica neuromuscular (Egr3) i transport de substàncies (Htr3a, Tnpo, Mlc1, Slc15a2, Slc6a4), mostraria un alentiment de la regeneració nerviosa. En animals control ICR a les 4 setmanes després d’una lesió en el nervi ciàtic, la sobreregulació de gens implicats en reorganització o remodelació de la matriu extracel·lular (MEC) i del microambient local a la part lesionada (Mmp16, Col18a1, Col3a1, Col5a2, Loxl2) i d’adhesió cel·lular (Lmo7, Flrt3), i transport de substàncies (Cacna2d1, Slc15a3, Slc15a9, Slc6a4) i apoptosi (Phb, Comp), indicaria que hi ha regeneració i remodelació a la part lesionada. En animals transgènics (Tg-STZ) diabètics, els processos de reorganització i remodelació de la MEC quasi no estan sobrerregulats, excepte Mmp16 i Tgfbi amb un FC>2, el que confirmaria que la regeneració estaria retardada, i encara no s’haurien iniciat els fenòmens de remodelació local. En animals control ICR a les 4 setmanes després d’una lesió en el nervi ciàtic, hi ha sobrexpressió de gens relacionats amb el dolor neuropàtic (Npy, Npy2r, Gal) i en animals transgènics (Tg-STZ) no hi ha expressió de Npy, i el gen Gal està infrarregulat. Això podria estar relacionat amb la pèrdua de la sensibilitat al dolor que s’observa en els pacients amb PND. El coneixement dels gens i els processos biològics involucrats en la degeneració i regeneració nerviosa en ratolins sans ICR i en ratolins transgènics Tg-STZ diabètics, obre noves vies de recerca per aprofundir en el coneixement de la patogènia de la PND i per l’estudi de molècules diana en el tractament de la PND i del dolor neuropàtic.Diabetic neuropathy (PND) is the most frequent secondary complication of diabetes mellitus (DM). The most important contributors to reduction in the quality of life of patients with DM are neurological pain and foot ulcerations and, ultimately, non-traumatic amputation of the affected limb. The pathogenesis is multifactorial, there are structural changes in the peripheral nerves and current treatments remain largely symptomatic, non-specific and not uniformly effective. Transgenic diabetic RIP/INFβ mice when treated with low doses of streptozotocin (STZ) present with pancreatic β-cell destruction, mimicking autoimmune diabetes in humans. On the basis that mechanisms governing peripheral nerve degeneration and regeneration are controlled by dorsal root ganglion (GAD), the aim of this thesis was to characterize gene expression, and thus the biological processes that take place in the GAD of injured sciatic nerve, of transgenic RIP/INFβ mice treated with multiple low doses of STZ (Tg-STZ). Gene expression in GAD was studied in four different groups of mice: wild type ICR (ICR) as a wild type genetic background control; ICR treated with STZ (ICR-STZ); transgenic RIP/INFβ mice (Tg); and Tg mice treated with STZ (Tg-STZ). Four weeks after diabetes was established in Tg-STZ mice, a left sciatic nerve crush injury was performed in all groups, the right limb was left intact. Four weeks after sciatic nerve injury GAD samples were collected from both hind limbs. GAD RNA was extracted to perform microarray study (Affymetrix GeneChip® Mouse Genome 430 2.0 Array). Real time PCR (RT-qPCR) was performed to validate some of gene expression values obtained. Comparisons were made between the gene profiles from the different experimental conditions: 1) ICR-STZ injured vs ICR injured, 2) ICR injured vs ICR uninjured, 3) Tg-STZ uninjured vs ICR uninjured and 4) Tg-STZ injured vs ICR injured. Baseline samples were the uninjured limbs from ICR mice with a Fold Change value of 1. Once the compared profiles were obtained, upregulated and downregulated genes were analysed, as well as their related biological process, using different databases (Database for Annotation, Visulaization and Integrated Discovery; Center for Quantitaive Biology; Gene Home). The absence of differences in gene expression between injured limbs GAD from ICR-STZ mice vs ICR mice showed that STZ administration had no toxic effect in GAD. Four weeks after sciatic nerve injury, genes associated with nerve regeneration were still upregulated in ICR mice, particularly those involved in signalling (Gpr151, Nts), synaptic transmission (Npy), neuronal projection and axonal guidance (Sox11, Atf3, Tnc, Sema6a, Sprr1a, Gal). This finding indicates that the lesion was not completely resolved. Transgenic diabetic mice (Tg-STZ) showed downregulation of genes related to metabolic pathways (Vgf) and upregulation of genes implicated in carbohydrate (Car3) and lipid metabolism (Gdpd3), indicating that 8 weeks after DM onset, a sustained hyperglycaemia affects GAD. Transgenic diabetic mice (Tg-STZ) showed, after 8 weeks of sustained hyperglycaemia, downregulation of genes associated with nervous system development (Gal, Bdnf, Erg3), axonal regeneration (Sprr1a, Lingo1), synaptic transmission (Calb1, Snapin), signal transduction (Nts, Rgs2), potassium channel (Kcns1, Kcnq5, Knj13, Kcna6, Kcnt1) and apoptosis (Tfnaip, Ctsb, E2f1, Crh). Reflecting GAD degenerative changes due to PND. Four weeks after sciatic nerve injury, genes associated with nerve regeneration were still upregulated in diabetic transgenic mice (Tg-STZ) such as those involved in axon guidance (Foxd1), axonal regeneration (Sprr1a), neuronal projection (Mapk8), GAD neurons survival and neurite elongation (Gal, Sox11), or signalling (Npy1r, Igf1, Rgs18, Gpr151), synaptic transmission (Gabrb3, Neto1, CcKbr) and apoptosis (Crh). Suggesting ongoing posttraumatic GAD regeneration processes. Four weeks after sciatic nerve injury, several genes were downregulated in diabetic transgenic mice (Tg-STZ) involved in axogenesis (Nptx1, Cxcr4), neuromuscular synaptic transmission (Egr3) and transport (Htr3a, Tnpo, Mlc1, Slc15a2, Slc6a4), evidencing delayed nerve regeneration. Four weeks after sciatic nerve injury, genes associated with nerve regeneration were still upregulated in ICR mice, involved in extracellular matrix (MEC) reorganization and local microenvironment (Mmp16, Col18a1, Col3a1, Col5a2, Loxl2), cell adhesion (Lmo7, Flrt3), transport (Cacna2d1, Slc15a3, Slc15a9, Slc6a4) and apoptosis (Phb, Comp) indicating reorganization and regeneration of the injured site. Transgenic diabetic mice (Tg-STZ) only showed upregulation of 2 genes (Mmp16, Tgfbi) related to MEC reorganization. The remaining genes were barely upregulated. This finding is in accordance with a delayed regeneration, and with the fact that local remodelling is yet to be started. Four weeks after sciatic nerve injury, genes associated with neuropatic pain were upregulated in ICR mice (Npy, Npy2r, Gal). However, in diabetic transgenic mice Tg-STZ, no Npy expression was found and, moreover, Gal was downregulated. This finding could be related to PND loss of pain sensitivity. Our findings regarding genes and biological processes involved in nerve degeneration and regeneration mechanisms in ICR mice and in diabetic transgenic mice Tg-STZ lead to new research lines aimed to understanding PND pathogenesis and to the study of target therapeutic molecules for PND and diabetic pain

Anàlisi de l'expressió gènica en els ganglis de les arrels dorsals del nervi ciàtic en el ratolí transgènic rip/infβ, model de neuropatia diabètica

Diabetic neuropathy (PND) is the most frequent secondary complication of diabetes mellitus (DM). The most important contributors to reduction in the quality of life of patients with DM are neurological pain and foot ulcerations and, ultimately, non-traumatic amputation of the affected limb. The pathogenesis is multifactorial, there are structural changes in the peripheral nerves and current treatments remain largely symptomatic, non-specific and not uniformly effective. Transgenic diabetic RIP/INFβ mice when treated with low doses of streptozotocin (STZ) present with pancreatic β-cell destruction, mimicking autoimmune diabetes in humans. On the basis that mechanisms governing peripheral nerve degeneration and regeneration are controlled by dorsal root ganglion (GAD), the aim of this thesis was to characterize gene expression, and thus the biological processes that take place in the GAD of injured sciatic nerve, of transgenic RIP/INFβ mice treated with multiple low doses of STZ (Tg-STZ). Gene expression in GAD was studied in four different groups of mice: wild type ICR (ICR) as a wild type genetic background control; ICR treated with STZ (ICR-STZ); transgenic RIP/INFβ mice (Tg); and Tg mice treated with STZ (Tg-STZ). Four weeks after diabetes was established in Tg-STZ mice, a left sciatic nerve crush injury was performed in all groups, the right limb was left intact. Four weeks after sciatic nerve injury GAD samples were collected from both hind limbs. GAD RNA was extracted to perform microarray study (Affymetrix GeneChip® Mouse Genome 430 2.0 Array). Real time PCR (RT-qPCR) was performed to validate some of gene expression values obtained. Comparisons were made between the gene profiles from the different experimental conditions: 1) ICR-STZ injured vs ICR injured, 2) ICR injured vs ICR uninjured, 3) Tg-STZ uninjured vs ICR uninjured and 4) Tg-STZ injured vs ICR injured. Baseline samples were the uninjured limbs from ICR mice with a Fold Change value of 1. Once the compared profiles were obtained, upregulated and downregulated genes were analysed, as well as their related biological process, using different databases (Database for Annotation, Visulaization and Integrated Discovery; Center for Quantitaive Biology; Gene Home). The absence of differences in gene expression between injured limbs GAD from ICR-STZ mice vs ICR mice showed that STZ administration had no toxic effect in GAD. Four weeks after sciatic nerve injury, genes associated with nerve regeneration were still upregulated in ICR mice, particularly those involved in signalling (Gpr151, Nts), synaptic transmission (Npy), neuronal projection and axonal guidance (Sox11, Atf3, Tnc, Sema6a, Sprr1a, Gal). This finding indicates that the lesion was not completely resolved. Transgenic diabetic mice (Tg-STZ) showed downregulation of genes related to metabolic pathways (Vgf) and upregulation of genes implicated in carbohydrate (Car3) and lipid metabolism (Gdpd3), indicating that 8 weeks after DM onset, a sustained hyperglycaemia affects GAD. Transgenic diabetic mice (Tg-STZ) showed, after 8 weeks of sustained hyperglycaemia, downregulation of genes associated with nervous system development (Gal, Bdnf, Erg3), axonal regeneration (Sprr1a, Lingo1), synaptic transmission (Calb1, Snapin), signal transduction (Nts, Rgs2), potassium channel (Kcns1, Kcnq5, Knj13, Kcna6, Kcnt1) and apoptosis (Tfnaip, Ctsb, E2f1, Crh). Reflecting GAD degenerative changes due to PND. Four weeks after sciatic nerve injury, genes associated with nerve regeneration were still upregulated in diabetic transgenic mice (Tg-STZ) such as those involved in axon guidance (Foxd1), axonal regeneration (Sprr1a), neuronal projection (Mapk8), GAD neurons survival and neurite elongation (Gal, Sox11), or signalling (Npy1r, Igf1, Rgs18, Gpr151), synaptic transmission (Gabrb3, Neto1, CcKbr) and apoptosis (Crh). Suggesting ongoing posttraumatic GAD regeneration processes. Four weeks after sciatic nerve injury, several genes were downregulated in diabetic transgenic mice (Tg-STZ) involved in axogenesis (Nptx1, Cxcr4), neuromuscular synaptic transmission (Egr3) and transport (Htr3a, Tnpo, Mlc1, Slc15a2, Slc6a4), evidencing delayed nerve regeneration. Four weeks after sciatic nerve injury, genes associated with nerve regeneration were still upregulated in ICR mice, involved in extracellular matrix (MEC) reorganization and local microenvironment (Mmp16, Col18a1, Col3a1, Col5a2, Loxl2), cell adhesion (Lmo7, Flrt3), transport (Cacna2d1, Slc15a3, Slc15a9, Slc6a4) and apoptosis (Phb, Comp) indicating reorganization and regeneration of the injured site. Transgenic diabetic mice (Tg-STZ) only showed upregulation of 2 genes (Mmp16, Tgfbi) related to MEC reorganization. The remaining genes were barely upregulated. This finding is in accordance with a delayed regeneration, and with the fact that local remodelling is yet to be started. Four weeks after sciatic nerve injury, genes associated with neuropatic pain were upregulated in ICR mice (Npy, Npy2r, Gal). However, in diabetic transgenic mice Tg-STZ, no Npy expression was found and, moreover, Gal was downregulated. This finding could be related to PND loss of pain sensitivity. Our findings regarding genes and biological processes involved in nerve degeneration and regeneration mechanisms in ICR mice and in diabetic transgenic mice Tg-STZ lead to new research lines aimed to understanding PND pathogenesis and to the study of target therapeutic molecules for PND and diabetic pain

Pharmacokinetic Analysis of Omomyc Shows Lasting Structural Integrity and Long Terminal Half-Life in Tumor Tissue

MYC is an oncoprotein causally involved in the majority of human cancers and a most wanted target for cancer treatment. Omomyc is the best-characterized MYC dominant negative to date. In the last years, it has been developed into a therapeutic miniprotein for solid tumor treatment and recently reached clinical stage. However, since the in vivo stability of therapeutic proteins, especially within the tumor vicinity, can be affected by proteolytic degradation, the perception of Omomyc as a valid therapeutic agent has been often questioned. In this study, we used a mass spectrometry approach to evaluate the stability of Omomyc in tumor biopsies from murine xenografts following its intravenous administration. Our data strongly support that the integrity of the functional domains of Omomyc (DNA binding and dimerization region) remains preserved in the tumor tissue for at least 72 hours following administration and that the protein shows superior pharmacokinetics in the tumor compartment compared with blood serum