Development of a Liver Gene Therapy Strategy for Haemophilia B With Lentiviral Vectors

Lentiviral vectors (LVs) are attractive tools for liver gene therapy, by virtue of their ability to stably integrate in the genome of target cells and the absence of pre-existing humoral and cellular immunity against vector components in most humans. We have previously reported long-term phenotypic correction of haemophilia B and transgene-specific immune tolerance induction after a single intravenous administration of LVs in mice, provided that transgene expression is stringently targeted to hepatocytes. This is achieved by a combination of transcriptional control, mediated by a synthetic hepatocyte-specific promoter and post-transcriptional control obtained by including in the transgene sequences complementary to the haematopoietic-specific microRNA 142, which binds and targets for degradation any residual transgene mRNA expressed in antigen presenting cells of liver and spleen. We have now evaluated this gene therapy strategy in a large animal model. Our results show long-term canine Factor IX (FIX) expression up to 0.5-1% of normal levels and clinical improvement (almost complete prevention of spontaneous bleedings) in two haemophilia B dogs (>3.5 years cumulative follow up), with mild acute toxicity and without long-term adverse effects nor anti-transgene immune responses. The use of codon-optimised and hyper-functional FIX transgenes increased the potency of LVs (in the pharmacological meaning of efficacy per dose) >15-fold, allowing correction of the disease phenotype at low vector doses in mice, thus improving the therapeutic index of the gene therapy and prompting us to test this improvement in the next treated dog. We have investigated additional improvements in the potency of LVs for liver gene therapy, such as the use of the baculovirus envelope protein gp64, which improves hepatocyte targeting and LV particles resistance to complement-mediated inactivation. We performed a quantitative analysis of LV biodistribution within the liver cell populations and show that Kupffer cells uptake most vector genomes, despite being a small fraction of the total cells, and limit hepatocyte transduction at low administered LV doses. However, pre-treatment with a single dose of a clinically used proteasome inhibitor prior to LV administration reduces this trapping effect and increases hepatocyte transduction and therapeutic efficacy up to 3-fold in haemophilia B mice. We provide evidence that liver gene therapy can establish long-term FIX expression and immune tolerance in mice even in the presence of pre-existing anti-FIX antibody immunity. Since insertional mutagenesis is a concern for integrating vectors, we set out to explore the potential advantages of integrase-defective LVs (IDLVs) to express transgenes in the adult liver, in which hepatocytes turnover is slow. We show that, while not optimal for stable gene replacement therapy in their current design, IDLVs may represent a valuable strategy to induce stable antigen-specific tolerance by transient gene transfer and offer a treatment for immune-mediated diseases. On the other hand, since LV integration is preferable for efficient stable liver gene transfer, we stringently assessed the risk of oncogenesis associated to LV integration in ad hoc mouse models that are sensitised to develop hepatocellular carcinoma and found no detectable increase in carcinogenesis upon liver gene therapy with LVs. Overall our results position LVs as a promising platform for liver gene therapy that may well complement other available vectors to address the different challenges posed by the presentation of haemophilia and its complications in different patients and clinical conditions and may conceivably offer a therapeutic option for lysosomal and metabolic diseases

Gene transfer and genome editing for familial hypercholesterolemia

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease characterized by high circulating low-density lipoprotein (LDL) cholesterol. High circulating LDL cholesterol in FH is due to dysfunctional LDL receptors, and is mainly expressed by hepatocytes. Affected patients rapidly develop atherosclerosis, potentially leading to myocardial infarction and death within the third decade of life if left untreated. Here, we introduce the disease pathogenesis and available treatment options. We highlight different possible targets of therapeutic intervention. We then review different gene therapy strategies currently under development, which may become novel therapeutic options in the future, and discuss their advantages and disadvantages. Finally, we briefly outline the potential applications of some of these strategies for the more common acquired hypercholesterolemia disease

173. Insulin B9-23 LV-Driven Expression in Hepatocytes Combined With Suboptimal Dose of Anti-CD3 mAb Cures Type 1 Diabetes in NOD Mice

Type 1 diabetes (T1D) is an autoimmune disease resulting in complete destruction of insulin-producing pancreatic β cells. In T1D in human and in the non-obese diabetic (NOD) mouse, the spontaneous murine model of T1D, auto-reactive T cells target islet-associated antigens. Induction of antigen (Ag)-specific tolerance could cure Type 1 Diabetes (T1D) but it has not been achieved yet. We previously showed that lentiviral vector (LV)-mediated gene expression in hepatocytes induces active tolerance toward the encoded-Ag. Systemic administration of a single dose of Integrase competent (IC) or integrase defective (ID) LV.ET.InsB9-23.142T, enabling stable and transient expression of InsB9-23 in hepatocytes, respectively, arrests β cell destruction in NOD mice at advanced pre-diabetic stage by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). In the present study we tested the efficacy of hepatocytes-directed LV.ET. InsB9-23.142T gene transfer in protecting from disease progression at later stages and in reversing T1D.Treatment with LV.ET.InsB9-23.142T in NOD mice with glucose levels ranging from 200mg/mL to 250mg/mL blocked T1D progression in only 27% of the mice. Co-expression of the late auto-Ags-derived epitopes GAD206-220 and IGRP195-214 in hepatocytes did not improve the efficacy of LV.ET.InsB9-23.142T treatment. LV.ET.InsB9-23.142T treatment in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL did not result in reversion to normoglycemic levels in any of the treated mice.We next combined InsB9-23 gene transfer with anti-CD3 monoclonal antibody (mAb) treatment. Treatment with anti-CD3 mAb at optimal doses is able per se to reverse T1D in NOD mice. Therefore, we tested decreasing doses of anti-CD3 mAb in diabetic NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL to identify the sub-optimal dose unable to revert T1D. We found that a single administration of anti-CD3 mAb at 5μg instead of 10μg results was not effective. This sub-optimal dose of anti-CD3 mAb (1X 5μg) was administered together with LV.ET.InsB9-23.142T to NOD mice with blood glucose levels ranging from 250mg/mL to 300mg/mL. Results showed T1D reversal in 75% of ICLV-treated and 40% of the IDLV-treated mice. These data indicate that the LV.ET. InsB9-23.142T treatment combined with sub-optimal anti-CD3 mAb treatment is able to reverse overt diabetes

Bioactive and Structural Metabolites of Pseudomonas and Burkholderia Species Causal Agents of Cultivated Mushrooms Diseases1

Pseudomonas tolaasii, P. reactans and Burkholderia gladioli pv. agaricicola, are responsible of diseases on some species of cultivated mushrooms. The main bioactive metabolites produced by both Pseudomonas strains are the lipodepsipeptides (LDPs) tolaasin I and II and the so called White Line Inducing Principle (WLIP), respectively, LDPs which have been extensively studied for their role in the disease process and for their biological properties. In particular, their antimicrobial activity and the alteration of biological and model membranes (red blood cell and liposomes) was established. In the case of tolaasin I interaction with membranes was also related to the tridimensional structure in solution as determined by NMR combined with molecular dynamic calculation techniques. Recently, five news minor tolaasins, tolaasins A–E, were isolated from the culture filtrates of P. tolaasii and their chemical structure was determined by extensive use of NMR and MS spectroscopy. Furthermore, their antimicrobial activity was evaluated on target micro-organisms (fungi—including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.—chromista, yeast and bacteria). The Gram positive bacteria resulted the most sensible and a significant structure-activity relationships was apparent. The isolation and structure determination of bioactive metabolites produced by B. gladioli pv. agaricicola are still in progress but preliminary results indicate their peptide nature. Furthermore, the exopolysaccharide (EPS) from the culture filtrates of B. gladioli pv. agaricicola, as well as the O-chain and lipid A, from the lipopolysaccharide (LPS) of the three bacteria, were isolated and the structures determined

6. Targeted Genome Editing of Cell Lines for Improved and Scalable Production of Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. The manufacturing of clinical-grade LVs relies on transient transfection of vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic and antigenic class-I major histocompatibility complex (MHC-I) expressed on LV packaging cells and subsequently incorporated on the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of the MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. We generated B2M-negative cells devoid of surface-exposed MHC-I, which retain the ability to produce LVs. In order to insert the LV genome of interest in the packaging cell line, we performed site-specific integration in predetermined loci of the genome of these cells, chosen for robust expression, exploiting artificial nucleases and homology-directed repair. In several independent iterations of this process, we generated producer cell lines both for LV expressing marker genes and a therapeutic gene, i.e. coagulation factor IX (FIX), the gene mutated in hemophilia B. We show that these LV producer cells are stable in culture and can produce several liters of LV-containing conditioned medium. These LVs have comparable or only slightly lower infectious titer and specific infectivity than LVs produced by state-of-the-art transient transfection process and can transduce therapeutically relevant target cells, such as hematopoietic stem/progenitor cells and T lymphocytes to high efficiency. Moreover, we intravenously administered FIX-expressing LVs produced by the cell line to hemophilia B mice and established therapeutic levels of circulating FIX. These data indicate that site-specific integration is an efficient, rapid and reproducible method to generate LV producer cells, starting from a universal stable inducible LV packaging cell line. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the quality, safety and sustainability of LV production for clinical use

Probiotics May Improve Serum Folate Availability in Pregnant Women: A Pilot Study

BACKGROUND: Probiotics are living microorganisms that confer a health benefit when administered in adequate amounts. There is evidence in the current literature about the importance of probiotic use in pregnancy. The early supplementation of probiotics in the perinatal and postnatal periods seems to have a positive impact on the overall mother’s health and future health of infants. AIM: Our pilot study aimed to test the ability of specific probiotics strains in combination with the kiwi-unique enzyme actinidin to improve the availability of folic acid in 20 pregnant women. METHODS: We investigate 20 pregnant women in early (4–10 weeks) (6 patients), intermedium (11–20 weeks) (6 patients), and late (21–30 weeks) (8 patients) pregnancy. RESULTS: Our findings show that the tested formula promotes increased concentration of serum folate in women’s blood and contributes to the control of blood sugar and body weight, regardless of the gestational period (early, intermediate or late). CONCLUSIONS: Our data support the main results reported in the scientific literature about the importance of probiotics intake in pregnancy

28. Intravenous Administration of Lentiviral Vectors Expressing Hyperactive Factor IX Converts Severe Into Mild Hemophilia B in a Canine Model

Lentiviral vectors (LVs) are attractive vehicles for liver-directed gene therapy by virtue of their ability to stably integrate in the genome of target cells and the lack of pre-existing immunity against vector components in most humans. Over the past years, we have developed a LV platform that can achieve stable transgene expression in the liver, induce transgene-specific immune tolerance and establish correction of hemophilia in mouse models upon systemic administration. This LV is designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We then investigated the efficacy and safety profile of portal vein administration of LVs expressing wild-type, codon-optimized (c.o.) or c.o. and hyperactive factor IX (FIX) in a canine model of hemophilia B. We produced large-scale batches of LVs qualified for in vivo administration and treated adult hemophilia B dog by portal vein administration. We observed long-term stable reconstitution of canine FIX activity up to 1% of normal and significant amelioration of the clinical phenotype in 3 treated dogs (>9 years cumulative follow up). LV infusion was associated with transient signs of inflammation and mild hepatotoxicity, which could be abrogated by pretreatment with anti-inflammatory drugs. There was no detectable long-term toxicity or development of FIX inhibitors. In the perspective of clinical translation and to increase therapeutic efficacy, we next treated an 11-kg, hemophilia B dog by peripheral vein administration of LVs expressing the c.o. and hyperactive canine FIX at a 5-fold higher dose than those previously administered. At the current follow-up (3 months after gene therapy) FIX activity is 6-9% of normal. Intravenous LV administration, coupled with a 1-day anti-inflammatory and anti-histamine pre-treatment, induced mild and selflimiting leukopenia and elevation of aminotransferases. Treatment of more hemophilia B dogs is underway to confirm and extend these results. Overall, our studies, which suggest comparable efficacy of LV by both portal and peripheral vein administration, position LV-mediated liver gene therapy for further pre-clinical development and clinical translation. LVs may thus complement other available vectors to address some of the outstanding challenges posed by liver gene therapy of hemophilia and conceivably other diseases

756 stable amelioration of hemophilia b in dogs by intravenous administration of lentiviral vectors expressing hyper functional factor ix

Lentiviral vectors (LVs) are attractive vehicles for liver-directed gene therapy by virtue of their ability to stably integrate in the genome of target cells and the low prevalence of pre-existing immunity against HIV in humans. Over the past years, we have developed a LV platform that can achieve stable transgene expression in the liver, induce transgene-specific immune tolerance and establish correction of hemophilia in mouse models upon systemic administration. This LV is designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We then investigated the efficacy and safety profile of portal vein administration of LVs expressing canine factor IX (FIX) in a canine model of hemophilia B. We produced large-scale batches of LVs qualified for in vivo administration and treated adult hemophilia B dog by portal vein administration. We observed long-term stable reconstitution of canine FIX activity up to 1% of normal and significant amelioration of the clinical phenotype in 3 treated dogs with 6, 3.5 and 2.5 years of follow up. LV infusion was associated with transient signs of inflammatory response and mild hepatotoxicity, which could be abrogated by pretreatment with anti-inflammatory drugs. There was no detectable long-term toxicity or development of FIX inhibitors. In the perspective of clinical translation and to increase therapeutic efficacy, we next treated two 10-kg hemophilia B dogs by peripheral vein administration of LVs expressing a codon-optimized and hyperfunctional canine FIX at a 5-fold higher dose than those previously administered. Intravenous LV administration was well tolerated with mild and self-limiting elevation of aminotransferases in one dog. In the dog that reached more than 1 year of follow up FIX activity ranged between 4-8% of normal. Treatment of two more dogs at a higher dose is underway. Overall, our studies position LV-mediated liver gene therapy for further pre-clinical development and clinical translation. LVs may thus complement other available vectors to address some of the outstanding challenges posed by liver gene therapy of hemophilia and conceivably other diseases

286 genome editing of inducible cell lines for scalable production of improved lentiviral vectors for human gene therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. Current manufacturing of clinical-grade LVs mostly relies on transient transfection of plasmids expressing the multiple vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. These LV packaging cells are stable in culture even after single-cell cloning and can be scaled up to large volumes. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic class-I major histocompatibility complexes (MHC-I) expressed on LV packaging cells and incorporated in the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of all MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. The resulting B2M-negative cells were devoid of surface-exposed MHC-I and produced MHC-free LVs. These LVs retain their infectivity on all tested cells in vitro and efficiently transduced the mouse liver upon intravenous administration. Strikingly, the MHC-free LVs showed significantly reduced immunogenicity in a T-cell activation assay performed on human primary T cells co-cultured with syngeneic monocytes exposed to LV, from several (n=7) healthy donors. To reproducibly generate LV-producer cell lines from these cells, we insert the LV genome of interest in the AAVS1 locus, chosen for robust expression, exploiting engineered nucleases and homology-directed repair. By this strategy, we have obtained several independent producer cell lines for LVs that express marker or therapeutic genes and are devoid of plasmid DNA contamination. LVs produced by these cells reproducibly show titer and infectivity within the lower bound range of standard optimized transient transfection, and effectively transduce relevant target cells, such as hematopoietic stem/progenitor cells and T cells ex vivo and the mouse liver in vivo. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the yield, quality, safety and sustainability of LV production for clinical use

Efficacy of Sea Salt-Based Mouthwash and Xylitol in Improving Oral Hygiene among Adolescent Population: A Pilot Study

The scientific community has definitely demonstrated the importance of the use of mouthwash in daily oral hygiene. In our pilot study, we tested the effectiveness of a novel mouth rinse containing sea salt, xylitol, and lysozyme. Streptococcus mutans (S. mutans) growth, and plaque index in adolescent patients aged 14–17 years, were observed. The bacterial load was investigated by in vitro microbiological analysis; the plaque index was assessed through the O’Leary’s Plaque Control Record (PCR). The study has shown that the use of a sea salt-based mouthwash in daily oral hygiene reduces the bacterial levels of S. mutans (p < 0.01) linked to the combined action of xylitol and lysozyme, together with the action of sea salt. Our preliminary data confirm and improve the main results reported in the scientific literature on the importance of the use of xylitol, lysozyme, and sea salt in oral health