Fabio Russo

Long-term amelioration of feline Mucopolysaccharidosis VI after AAV-mediated liver gene transfer.

Mucopolysaccharidosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening. Gene transfer to a factory organ like liver may provide a lifetime source of secreted ARSB. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to 1 year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6 × 10(13) genome copies (gc)/kg) whereas delivery of AAV2/8 vector doses as low as 2 × 10(12) gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age at treatment, we observed: (i) clearance of GAG storage, (ii) improvement of long bone length, (iii) reduction of heart valve thickness, and (iv) improvement in spontaneous mobility. Thus, AAV2/ 8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.

Role of the dorsal hippocampus in object memory load

The dorsal hippocampus is crucial for mammalian spatial memory, but its exact role in item memory is still hotly debated. Recent evidence in humans suggested that the hippocampus might be selectively involved in item short-term memory to deal with an increasing memory load. In this study, we sought to test this hypothesis. To this aim we developed a novel behavioral procedure to study object memory load in mice by progressively increasing the stimulus set size in the spontaneous object recognition task. Using this procedure, we demonstrated that naive mice have a memory span, which is the number of elements they can remember for a short-time interval, of about six objects. Then, we showed that excitotoxic selective lesions of the dorsal hippocampus did not impair novel object discrimination in the condition of low memory load. In contrast, the same lesion impaired novel object discrimination in the high memory load condition, and reduced the object memory span to four objects. These results have important heuristic and clinical implications because they open new perspective toward the understanding of the role of the hippocampus in item memory and in memory span deficits occurring in human pathologies, such as Alzheimers disease and schizophrenia.

Different serum enzyme levels are required to rescue the various systemic features of the mucopolysaccharidoses.

Mucopolysaccharidoses (MPSs) are lysosomal storage disorders characterized by progressive accumulation of glycosaminoglycans (GAGs) in various tissues. Enzyme replacement therapy (ERT) for several MPSs is available to date. However, the efficacy of ERT is limited, in particular in compartments such as bone, cartilage, the brain, and the eyes. We selected a rodent model of an MPS, with no central nervous system storage, to study the impact, on systemic features of the disease, of various stable levels of exogenous enzymes produced by adeno-associated viral vector (AAV)-mediated liver gene transfer. Low levels (6% of normal) of circulating enzyme were enough to reduce storage and inflammation in the visceral organs and to ameliorate skull abnormalities; intermediate levels (11% of normal) were required to reduce urinary GAG excretion; and high levels (>or=50% of normal) rescued abnormalities of the long bones and motor activity. These data will be instrumental to design appropriate clinical protocols based on either enzyme or gene replacement therapy for MPS and to predict their impact on the pathological features of MPS.

Worsening of cardiomyopathy using deflazacort in an animal model rescued by gene therapy.

We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients.

Gradient COUP-TFI Expression Is Required for Functional Organization of the Hippocampal Septo-Temporal Longitudinal Axis

Abstract The hippocampus (HP), a medial cortical structure, is subdivided into a distinct dorsal (septal) and ventral (temporal) portion, which is separated by an intermediate region lying on a longitudinal curvature. While the dorsal portion is more dedicated to spatial navigation and memory, the most ventral part processes emotional information. Genetic factors expressed in gradient during development seem to control the size and correct positioning of the HP along its longitudinal axis; however, their roles in regulating differential growth and in supporting its anatomical and functional dissociation remain unexplored. Here, we challenge the in vivo function of the nuclear receptor COUP‐TFI (chicken ovalbumin upstream promoter transcription factor 1) in controlling the hippocampal, anatomical, and functional properties along its longitudinal axis. Loss of cortical COUP‐TFI function results in a dysmorphic HP with altered shape, volume, and connectivity, particularly in its dorsal and intermediate regions. Notably, topographic inputs from the entorhinal cortex are strongly impaired in the dorsal portion of COUP‐TFI mutants. These severe morphological changes are associated with selective spatial learning and memory impairment. These findings identify a novel transcriptional regulator required in the functional organization along the hippocampal septo‐temporal axis supporting a genetic basis of the hippocampal volumetric growth with its final shape, circuit, and type of memory function.

Sensory-motor behavioral characterization of an animal model of Maroteaux-Lamy syndrome (or Mucopolysaccharidosis VI)

Maroteaux-Lamy disease, also known as mucopolysaccharidosis (MPS) VI, is an MPS disorder caused by mutations in the ARSB gene encoding for the lysosomal enzyme arysulfatase B (ARSB). Deficient ARSB activity leads to lysosomal accumulation of dermatan sulfate in a wide range of tissues and organs. There are various animal models of MPS VI that have been well characterized from a biochemical and morphological point of view. In this study, we report the sensory-motor characterization of MPS VI rats carrying homozygous null ARSB mutations. We show that adult MPS VI rats are specifically impaired in vertical activity and motor endurance. All together, these data are consistent with biochemical findings that show a major impairment in connective tissues, such as joints and bones. The behavioral abnormalities of MPS VI rats represent fundamental endpoints for studies aimed at testing the pre-clinical safety and efficacy of novel therapeutic approaches for MPS VI.

51. LV. InsB9-23/Anti-CD3 mAb Inhibits Recurrence of Autoimmunity in NOD Mice After Islet Transplants

Type 1 diabetes (T1D) is an autoimmune disease resulting in complete destruction of insulin-producing pancreatic beta-cells, which is mediated by auto-reactive T cells. In T1D in human and in the non-obese diabetic (NOD) mouse, the spontaneous murine model of T1D, induction of tolerance to beta-cell associated antigens represents a potential curative option. We previously showed that systemic administration of a single dose of integrase-competent (IC) or integrase-defective (ID) LV.ET. InsB9-23.142T (LV.InsB), enabling stable or transient expression of InsB9-23 in hepatocytes, arrests beta-cell destruction in NOD mice at advanced pre-diabetic stage. In these mice stable normoglycemia is maintained long term and InsB9-23-specific FoxP3+ T regulatory cells (Tregs) are generated. Moreover, LV. InsB in combination with a suboptimal dose (1X 5µg) of anti-CD3 mAb reverts overt diabetes and preserves residual beta-cell mass. In the present study we tested the efficacy of the LV.InsB/anti-CD3 combination therapy to inhibit recurrence of autoimmunity and maintain insulin independence after pancreatic islets transplants. Pancreatic islets isolated from NOD-scid donor mice were transplanted under the kidney capsule of diabetic NOD mice with blood glucose levels >350mg/dL. Successfully transplanted mice (normoglycemic: blood glucose levels ~100 mg/dL) were treated with LV.InsB/anti-CD3; anti-CD3 mAb (1X 5µg) alone or left untreated as controls. LV.InsB/anti-CD3 combination therapy allowed stable normoglycemia up to 250 days whereas in mice receiving anti-CD3 mAb alone or untreated controls recurrence of diabetogenic responses cleared transplanted islets in 2 weeks. Autoreactive T cells were still present in the spleen of transplanted mice treated with LV.InsB/anti-CD3, but the frequency of FoxP3+ Tregs within the CD4+ T cells in renal (RLN) and pancreatic (PLN) lymph nodes significantly increased, as shown by phenotypic analysis. These data show that LV. InsB/anti-CD3 treatment induces active suppression of autoimmune responses in long-term normoglycemic transplanted NOD mice. Ag-specific FoxP3+ Tregs accumulating in the PLN and RLN suppress effector T cells present in the target tissue. This mode of action is similar to what we previously described after LV. InsB treatment in a model of autoimmunity (Akbarpour M. et al. Science TM. 2015). Further studies are currently ongoing to evaluate the relative contribution of endogenous and exogenous beta-cell mass to insulin independence and to investigate the efficacy of the LV.InsB/anti-CD3 treatment after transplant of allogeneic pancreatic islets. The definition of novel gene therapy strategies to induce Ag-specific tolerance that at the same time control autoimmunity and transplant rejection would represent a major step toward the cure of type 1 diabetes.

637. Targeting of Myeloid Leukemia by IL-10-Engineered Human CD4+ Tr1 Cells

T regulatory type 1 (Tr1) cells, characterized by the co-expression of CD49b and LAG-3 and the ability to secrete high amounts of IL-10, control immune responses by IL-10 and TGF-beta production and by killing of myeloid cells via a Granzyme B-dependent mechanism. Tr1 cells are induced in vitro in the presence of recombinant human IL-10 or tolerogenic dendritic cells secreting high amounts of IL-10 (DC-10). Proof-of-principle clinical trials suggest that Tr1 cells can modulate Graft-versus Host Disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity or their effects of Graft versus Leukemia is largely unknown. We previously showed that enforced IL-10 expression converts human CD4+ T cells into Tr1-like (CD4IL-10) cells that suppress effector T cells in vitro and prevent xenogeneic-GvHD in humanized models. We now demonstrate that these CD4IL-10 cells selectively kill myeloid cell lines and myeloid blasts in vitro in HLA-class I-dependent but antigen-independent manner. Moreover, cytotoxic activity of CD4IL-10 cells is Granzyme B-dependent, is specific for CD13+ cells, and requires CD54 and CD112 expression on target cell lines or primary leukemic blast. Adoptive transfer of CD4IL-10 cells in humanized models mediates direct anti-leukemic activity, and does not compromise the anti-leukemic effect of allogeneic T cells while inhibits xeno-GvHD. These findings provide a strong rationale for designing personalized immunotherapy approaches using CD4IL-10 cells after allo-HSCT to cure myeloid malignancies.

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.