Joel Montane

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency.

The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, second-generation CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.

Reversal of Type 1 Diabetes by Engineering a Glucose Sensor in Skeletal Muscle

Type 1 diabetic patients develop severe secondary complications because insulin treatment does not guarantee normoglycemia. Thus, efficient regulation of glucose homeostasis is a major challenge in diabetes therapy. Skeletal muscle is the most important tissue for glucose disposal after a meal. However, the lack of insulin during diabetes impairs glucose uptake. To increase glucose removal from blood, skeletal muscle of transgenic mice was engineered both to produce basal levels of insulin and to express the liver enzyme glucokinase. After streptozotozin (STZ) administration of double-transgenic mice, a synergic action in skeletal muscle between the insulin produced and the increased glucose phosphorylation by glucokinase was established, preventing hyperglycemia and metabolic alterations. These findings suggested that insulin and glucokinase might be expressed in skeletal muscle, using adeno-associated viral 1 (AAV1) vectors as a new gene therapy approach for diabetes. AAV1-Ins+GK–treated diabetic mice restored and maintained normoglycemia in fed and fasted conditions for >4 months after STZ administration. Furthermore, these mice showed normalization of metabolic parameters, glucose tolerance, and food and fluid intake. Therefore, the joint action of basal insulin production and glucokinase activity may generate a “glucose sensor” in skeletal muscle that allows proper regulation of glycemia in diabetic animals and thus prevents secondary complications.

Treatment of Diabetes and Long-Term Survival After Insulin and Glucokinase Gene Therapy

Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a “glucose sensor” in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.

Molecular signature of the immune and tissue response to non-coding plasmid DNA in skeletal muscle after electrotransfer

Electrotransfer of plasmid DNA in skeletal muscle is a common non-viral delivery method for both therapeutic genes and DNA vaccines. Yet, despite the similar approaches, an immune response is detrimental in gene therapy, but desirable for vaccines. However, the full nature of the immune and tissue responses to nucleic acids and electrotransfer in skeletal muscle has not been addressed. Here we used microarray analysis, fluorescence-activated cell sorting and quantitative polymerase chain reaction to obtain the molecular and cellular signature of the tissue and immune response to electrotransfer of saline and non-coding plasmid DNA. Saline electrotransfer resulted in limited infiltration and induction of a moderate damage–repair gene expression pattern not involving innate immune activation. However, plasmid electrotransfer augmented expression of the same genes in addition to inducing a strong innate immune response associated with pro-inflammatory infiltration. In particular, the inflammasome, Toll-like receptor 9 and other pattern recognition receptors able to respond to cytoplasmic DNA were upregulated. Several key differences in the nature of the inflammatory infiltrate and the kinetics of gene expression were also identified when comparing electrotransfer of conventional and CpG-free plasmids. Our data provide insights into the mechanisms of DNA detection and response in muscle that has relevance for non-viral gene therapy and DNA vaccination.

Stress-Induced MicroRNA-708 Impairs β-Cell Function and Growth

The pancreatic β-cell transcriptome is highly sensitive to external signals such as glucose oscillations and stress cues. MicroRNAs (miRNAs) have emerged as key factors in gene expression regulation. Here, we aimed to identify miRNAs that are modulated by glucose in mouse pancreatic islets. We identified miR-708 as the most upregulated miRNA in islets cultured at low glucose concentrations, a setting that triggers a strong stress response. miR-708 was also potently upregulated by triggering endoplasmic reticulum (ER) stress with thapsigargin and in islets of ob/ob mice. Low-glucose induction of miR-708 was blocked by treatment with the chemical chaperone 4-phenylbutyrate, uncovering the involvement of ER stress in this response. An integrative analysis identified neuronatin (Nnat) as a potential glucose-regulated target of miR-708. Indeed, Nnat expression was inversely correlated with miR-708 in islets cultured at different glucose concentrations and in ob/ob mouse islets and was reduced after miR-708 overexpression. Consistent with the role of Nnat in the secretory function of β-cells, miR-708 overexpression impaired glucose-stimulated insulin secretion (GSIS), which was recovered by NNAT overexpression. Moreover, miR-708 inhibition recovered GSIS in islets cultured at low glucose. Finally, miR-708 overexpression suppressed β-cell proliferation and induced β-cell apoptosis. Collectively, our results provide a novel mechanism of glucose regulation of β-cell function and growth by repressing stress-induced miR-708.

Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment

Human islet amyloid polypeptide (hIAPP) aggregation is associated with β‐cell dysfunction and death in type 2 diabetes (T2D). we aimed to determine whether in vivo treatment with chemical chaperone 4‐phenylbutyrate (PBA) ameliorates hIAPP‐induced β‐cell dysfunction and islet amyloid formation. Oral administration of PBA in hIAPP transgenic (hIAPP Tg) mice expressing hIAPP in pancreatic β cells counteracted impaired glucose homeostasis and restored glucose‐stimulated insulin secretion. Moreover, PBA treatment almost completely prevented the transcriptomic alterations observed in hIAPP Tg islets, including the induction of genes related to inflammation. PBA also increased β‐cell viability and improved insulin secretion in hIAPP Tg islets cultured under glucolipotoxic conditions. Strikingly, PBA not only prevented but even reversed islet amyloid deposition, pointing to a direct effect of PBA on hIAPP. This was supported by in silico calculations uncovering potential binding sites of PBA to monomeric, dimeric, and pentameric fibrillar structures, and by in vitro assays showing inhibition of hIAPP fibril formation by PBA. Collectively, these results uncover a novel beneficial effect of PBA on glucose homeostasis by restoring β‐cell function and preventing amyloid formation in mice expressing hIAPP in β cells, highlighting the therapeutic potential of PBA for the treatment of T2D.—Montane, J., de Pablo, S., Castaño, C., Rodríguez‐Comas, J., Cadavez, L., Obach, M., Visa, M., Alcarraz‐Vizán, G., Sanchez‐Martinez, M., Nonell‐Canals, A., Parrizas, M., Ser‐vitja, J.‐M., Novials, A. Amyloid‐induced β‐cell dysfunction and islet inflammation are ameliorated by 4‐phenylbutyrate (PBA) treatment. FASEB J. 31, 5296–5306 (2017). www.fasebj.org

BACE2 suppression promotes β-cell survival and function in a model of type 2 diabetes induced by human islet amyloid polypeptide overexpression

BACE2 (β-site APP-cleaving enzyme 2) is a protease expressed in the brain, but also in the pancreas, where it seems to play a physiological role. Amyloidogenic diseases, including Alzheimer’s disease and type 2 diabetes (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. In T2D, islet amyloid polypeptide (IAPP) deposits have been shown to be a pathogenic key feature of the disease. The aim of the present study was to investigate the effect of BACE2 modulation on β-cell alterations in a mouse model of T2D induced by IAPP overexpression. Heterozygous mice carrying the human transcript of IAPP (hIAPP-Tg) were used as a model to study the deleterious effects of IAPP upon β-cell function. These animals showed glucose intolerance and impaired insulin secretion. When crossed with BACE2-deficient mice, the animals presented a significant improvement in glucose tolerance accompanied with an enhanced insulin secretion, as compared to hIAPP-Tg mice. BACE2 deficiency also partially reverted gene expression changes observed in islets from hIAPP-Tg mice, including a set of genes related to inflammation. Moreover, homozygous hIAPP mice presented a severe hyperglycemia and a high lethality rate from 8 weeks onwards due to a massive destruction of β-cell mass. This process was significantly reduced when crossed with the BACE2-KO model, improving the survival rate of the animals. Altogether, the absence of BACE2 ameliorates glucose tolerance defects induced by IAPP overexpression in the β-cell and promotes β-cell survival. Thus, targeting BACE2 may represent a promising therapeutic strategy to improve β-cell function in T2D.

The Role of Human IAPP in Stress and Inflammatory Processes in Type 2 Diabetes

Understanding the mechanisms regulating whole-body glucose homeostasis is important in order to understand what happens in a disease such as type 2 diabetes (T2D). Insulin resistance, inflammation, dysfunction of islet β-cells, and the presence of amyloid deposits in the pancreas are some of the major causes involved in the process of β-cell deterioration. The unique peptide constituent of amyloid deposits, human islet amyloid polypeptide (hIAPP), is capable of inducing endoplasmic reticulum (ER) stress and the resulting unfolded-protein response activation. Additionally, hIAPP has been shown to induce interleukin-1β expression, the main cytokine involved in inflamma‐ tion and T2D causing inflammation and eventually, inducing apoptosis. Nevertheless, the mechanisms behind the process of hIAPP aggregation and amyloid formation are still unknown. In this chapter, we describe the different mechanisms by which hIAPP induces ER stress and inflammation. This should open the door for designing therapeutic interventions aimed at modulating the immune system and the ER stress response.

155. Non-viral gene therapy for type 1 diabetes by electrotransfer of insulin and glucokinase genes to skeletal muscle

Type 1 diabetes patients depend upon insulin replacement therapy. However, glycemic control is not always achieved and the resulting chronic hyperglycemia leads to microvascular, macrovascular and neurological complications. Therefore, there is a need for new treatment strategies for diabetes mellitus that would permit tight glucose regulation. Transgenic mice expressing insulin in skeletal muscle counteract type 1 diabetic alterations which indicates that muscle cells constitutively secreting low insulin levels could be used in gene therapy for diabetes. Furthermore, expression of glucokinase in skeletal muscle of transgenic mice reduced diabetic hyperglycemia without inducing hypoglycemia. However neither of these two transgenic mice showed complete reversion of the diabetic phenotype. Here, we examined the effect of co-expression of insulin and glucokinase in skeletal muscle on diabetic alterations and glycemia. Double transgenic mice expressing both insulin and glucokinase in skeletal muscle and skeletal muscle from diabetic mice electrotransferred with insulin and glucokinase genes were obtained. Blood glucose levels, skeletal muscle glucose uptake, glucose tolerance and insulin sensitivity were analyzed after streptozotozin (Stz) treatment. Double transgenic and electrotransferred mice counteracted hyperglycemia and restored fluid and food intake after treatment with Stz. In contrast, control mice presented polydipsia and polyphagia and developed severe diabetes. No hypogluycemia was detected in the Stz-treated mice expressing both insulin and glucokinase genes. In addition, these mice normalised both hepatic and skeletal muscle glucose metabolism and showed increased glucose disposal after an intraperitoneal glucose tolerance test. These results suggest that secretion of basal levels of insulin, in conjunction with increased glucose uptake by the skeletal muscle, might permit tight regulation of glycemia. Insulin and glucokinase genes cooperated by increasing glucose transport into the muscle cell and phosphorylation. Thus, this study provides evidence of a feasible non-viral gene therapy approach for type 1 diabetes involving the cooperative action of two genes, glucokinase and insulin, which leads to tight regulation of glucose homeostasis.