FIELD OF THE INVENTION

The invention relates to a pharmaceutical composition for use in preventing and/or treating diabetes, said composition comprising a modulator of the Glis3-Manf pathway and a targeting moiety specific for a tissue or organ of a subject, such as the pancreas. Also provided are viral vectors and methods for preventing and/or treating diabetes.

BACKGROUND OF THE INVENTION

Diabetes is a disease of dysfunctional glucose regulation. The predominant forms of diabetes are type 1 diabetes (T1 D) and type 2 diabetes (T2D), although there are a diverse set of additional forms, which include aspects of one or both diseases. In T1 D, disease is initiated and caused by inheritance of an adaptive immune system that is predisposed to responding beta-cell antigens, most notably to insulin itself (Roizen et al. (2015) Curr Diab Rep 15, 102). However, despite autoimmunity being effectively established by three years of age in over 80% of cases, most patients are diagnosed many years, or even decades, after establishment of persistent anti-islet autoimmunity (Bonifacio et al. (2017) Diabetologia 60, 35-38. There have been over 35 years of failed immunotherapeutic trials aimed at stopping the autoimmune response in T1 D suggesting that these final stages, after the initial autoimmunity, may not solely be autoimmune in nature but may also be due to an intrinsic beta-cell vulnerability or fragility to cell death.

In contrast to T1 D, T2D is not an autoimmune disease. In the earliest pre-diabetic stages of the disease, normally insulin-responsive cells (such as hepatocytes) become resistant to insulin, with reduced signalling through the insulin receptor. The boundary between prediabetes and early-stage T2D is ill-defined (Heianza, Y. et al. (2012) Diabet Med 29, e279- 285), with pancreatic beta-cells initially able to compensate for insulin resistance by increasing the amount and duration of insulin secretion. Many patients remain in a grey area of diagnosis at this stage, where diet modification and anti-diabetogenic drugs are sufficient to avoid the prolonged hyper-glycemia that is the pathological outcome of untreated T2D. However, a sizable subset of early-stage T2D patients go on to develop a loss of beta-cell mass. A critical inflection point in the disease process is the point at which insulin levels (having escalated with increasing insulin resistance) start declining, with the loss in beta-cell numbers greater than the compensatory capacity of the remaining cells (Cnop et al. (2005) Diabetes 54 Suppl 2, S97-107; Butler et al. (2003) Diabetes 52, 102-110). In this late-stage of T2D, insulin administration is required, but, due to the pre-existing insulin resistance, outcomes are poor (Munnee et al. (2016) Medicine (Baltimore) 95, e3006). The mechanistic basis of beta-cell decline during the early-to-late stage transition is highly controversial, with convincing arguments being put forward as to the role of glucose toxicity (Maedler et al. (2001) Diabetes 50, 1683-1690), endogenous beta-cell stress due to excessive insulin production (Back and Kaufman (2012) Annu Rev Biochem 81 , 767-793), or inflammation-mediated destruction (Zhou et al. (2010) Nat Immunol 11 , 136-140), among other plausible hypotheses.

The concept that primary beta-cell defects may lie at the heart of susceptibility to both T1 D and T2D has been proposed by the present inventors (Dooley et al. (2016) Nature genetics 48, 519-527; Liston et al. (2017) Trends Mol Med 23(2), 181-194; and others (Ikegami et al. (2004) ILAR J 45, 268-277; Wilkin et al. (2016) Diabetes, obesity & metabolism 18, 3-5; Nogueira et al. PLoS Genet 9, e1003532), with various iterations and degrees of emphasis on the differences or similarities between T1 D and T2D. The beta-cell fragility model can be defined as one where variation in the intrinsic fragility or robustness of beta-cells contributes to the development of diabetes. Just as susceptibility to autoimmunity and insulin resistance varies across individuals, due to genetic and environmental influences, beta-cells vary across individuals in their ability to survive autoimmune or metabolic insults (Liston et al. (2017), supra). The presence of “fragile” beta-cells in an individual would sensitize for diabetes, either T1 D, for individuals with autoimmune susceptibility, or T2D, for individuals with metabolic stress. By contrast, individuals with “robust” beta-cells would be more likely to remain at a pre- clinical stage with delayed diabetes development. Under this model, increasing the robustness of beta-cells would be an effective therapeutic strategy for individuals at high risk for either T1 D or T2D, or to prevent further progression of T2D.

There is therefore a great unmet need for a therapy for diabetes, such as a targeted therapy which may be used to treat/relieve diabetes and related disorders.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a pharmaceutical composition comprising a modulator of the Glis3-Manf pathway and a targeting moiety specific for a tissue or organ of a subject for use in preventing and/or treating diabetes.

According to a further aspect of the invention, there is provided an adeno-associated virus (AAV) vector which encodes:

(a) a modulator of the Glis3-Manf pathway; and

(b) a tissue or organ specific promoter. According to a further aspect of the invention, there is provided a method of preventing and/or treating diabetes in a subject in need thereof, said method comprising administering to the subject the pharmaceutical composition or the vector as defined herein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Gene delivery of Manf to beta-cells of the pancreatic islets averts type 1 diabetes development. NOD mice were treated at 10 weeks of age with AAV-insManf or AAV-insGFP, and followed for diabetes development (n=13, 12).

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided a pharmaceutical composition comprising a modulator of the Glis3-Manf pathway and a targeting moiety specific for a tissue or organ of a subject for use in preventing and/or treating diabetes.

References herein to “modulator of the Glis3-Manf pathway” refer to any component which modulates (i.e. inhibits, agonises, antagonises etc) the Glis3-Manf pathway. In one embodiment, the modulator of the Glis3-Manf pathway is G I is3. Giis3 (GUS Family Zinc Finger 3) is a member of ths GU-similar zinc finger protein family and encodes a nuclear protein with five C2H2-type zinc finger domains. This protein functions as both a repressor and activator of transcription and is specifically involved in the development of pancreatic beta cells, the thyroid, eye, liver and kidney. Mutations in this gene have been associated with neonatal diabetes and congenital hypothyroidism (NDH). Alternatively spliced variants that encode different protein isoforms have been described but the full-length nature of only two have been determined.

In an alternative embodiment, the modulator of the Glis3-Manf pathway is Manf. Mesencephalic astrocyte-derived neurotrophic factor (Manf) is a small protein with a molecular mass of 18 kDa. It contains an amino-terminal signal peptide that directs it to the endoplasmic reticulum (ER) and, when cleaved, results in a mature protein that can be secreted.

References herein to the phrase “in a tissue or organ” refer to a discrete location in the subject such as in a particular tissue or organ. It will be appreciated that such terms do not relate to wherein an effect is produced systemically or outside of the tissue or organ of interest, or wherein a cell type or cell population not located in the tissue or organ of interest is affected. Tissues or organs as defined herein comprise a discrete location of the body or of an organism. For example, the tissue or organ may comprise a compartment of the body. In one embodiment, the tissue or organ is the pancreas. In a further embodiment, the tissue or organ is the pancreatic islets. In a yet further embodiment the tissue or organ is the beta cells of the pancreatic islets.

Support for the beta-cell fragility hypothesis comes from disease models that allow dissection of beta-cell-intrinsic function. The primary animal model of T1 D is the non-obese diabetic (NOD) mouse, which hosts a large set of genetic polymorphisms increasing susceptibility to anti-islet autoimmunity (Wicker et al. (2005) J Autoimmun 25 Suppl, 29-33). I ntriguingly, the NOD diabetes-associated loci overlap with diabetes-associated from the related T2D mouse strain, Nagoya-Shibata-Yasuda (NSY) mice (Ikegami et al. (2004), supra), raising the possibility of a shared genetic predisposition. The laboratory of the present inventors performed a molecular dissection of NOD genetic control over beta cell viability and found strong evidence that the shared component of diabetes susceptibility may be dependent on beta-cell fragility. NOD mice possess variants in Glis3 that have beta-cell-intrinsic functions, rendering the beta-cells highly susceptible to apoptosis (Dooley et al. (2016), supra). When NOD mice are exposed to beta-cell stressors that mimic the compensatory insulin overproduction observed in T2D, this beta-cell fragility is sufficient to tip the mice into overt diabetes, while the same stressors remain sub-clinical in mice with robust beta-cells (Dooley et al. (2016), supra).

The molecular mechanism by which the NOD Glis3 variant enhances beta-cell susceptibility to apoptosis appears to be via reduced upregulation of Manf, an obligate pro-survival factor for beta-cells (Lindahl et al. (2014) Cell reports 7, 366-375). The present inventors sought to increase the robustness of the beta-cells of the pancreas by correcting the defective upregulation of Manf present in NOD mice. Using an AAV-based gene delivery system, the present inventors drove expression of Manf in the beta-cells of NOD mice using the insulin promoter. Surprisingly, gene delivery of Manf substantially lowered the rate of diabetes development in treated mice, proof-of-principle that correcting beta cell fragility can avert clinical diabetes progression (see Example 1 and Figure 1).

In one embodiment, the method of preventing and/or treating diabetes comprises administration of the modulator of the Glis3-Manf pathway, such as tissue- or organ-specific expression of said modulator of the Glis3-Manf pathway in said tissue or organ of the subject.

In one embodiment, tissue- or organ-specific expression of the modulator of the Glis3-Manf pathway is driven by a tissue- or organ-specific promoter. In a further embodiment, the tissue- or organ-specific promoter is a pancreas specific promoter. In a yet further embodiment, the pancreas specific promoter is an insulin promoter.

In one embodiment, administration of the modulator of the Glis3-Manf pathway or tissue- or organ-specific expression of the modulator of the Glis3-Manf pathway in said tissue or organ comprises an exogenous encoding sequence of the modulator of the Glis3-Manf pathway.

Adeno-Associated Virus (AAV) Vectors

In one embodiment, the targeting moiety specific for the tissue or organ of the subject is a virus or viral vector as defined herein. In a further embodiment, said virus or viral vector specifically targets or infects the tissue or organ of interest or specifically targets or infects cells of the tissue or organ of interest (i.e. the pancreas). Thus according to this embodiment, said targeting moiety specific for the tissue or organ of interest which is a virus or viral vector does not target or infect cells in other tissues or organs other than the tissue or organ of interest, or target or infect cells which make up a tissue or organ other than the tissue or organ of interest. In a further embodiment, the viral vector is an adeno-associated virus (AAV) vector which specifically targets or infects the tissue or organ. In a yet further embodiment, the adeno- associated virus (AAV) vector is an AAV8 adeno-associated virus (AAV) vector.

According to a further aspect of the invention there is provided an adeno-associated virus (AAV) vector which encodes:

(a) a modulator of the Glis3-Manf pathway; and

(b) a tissue or organ specific promoter.

It will be appreciated that the modulator of the Glis3-Manf pathway is as defined herein and includes for example Glis-3 or Manf, such as Manf.

It will be appreciated that the tissue or organ specific promoter is as defined herein and includes a pancreas specific promoter, such as an insulin promoter.

Pharmaceutical Compositions

According to some embodiments, the pharmaceutical composition, in addition to the modulator of the Glis3-Manf pathway and the tissue or organ specific virus or viral vector as defined herein, further comprises one or more pharmaceutically acceptable excipients.

Generally, the present pharmaceutical compositions will be utilised with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a composition comprising the targeting moiety specific for a tissue or organ as defined herein in a discrete location (e.g. within a tissue or organ of interest), may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatine and alginates. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16 ^th Edition).

Therapeutic Uses and Methods

It will be appreciated from the disclosures presented herein that the methods of treatment described herein will find particular utility in the treatment and/or amelioration of diseases or disorders mediated by the Glis3-Manf pathway, such as diabetes.

Thus, according to a further aspect of the invention, there is provided a method of preventing and/or treating diabetes in a subject in need thereof, said method comprising administering to the subject the pharmaceutical composition or the vector as defined herein.

Diabetes mellitus, commonly known as diabetes, is a group of metabolic disorders characterized by a high blood sugar level over a prolonged period of time. Examples of diabetes include Type 1 diabetes (T1 D), Type 2 diabetes (T2D) and gestational diabetes. In one embodiment, said diabetes is Type 1 diabetes (T1 D). In an alternative embodiment, said diabetes is Type 2 diabetes (T2D).

It will be appreciated that references herein to diabetes also include diabetic related disorders. Such diabetic related disorders include celiac disease, thyroid disease, polycystic ovary syndrome, diabetes insipidus, necrobiosis lipoidica diabeticorum, mastopathy, muscular conditions including limited joint mobility, frozen shoulder, Dupuytren’s contracture, trigger finger, carpal tunnel syndrome, dental problems, haemochromatosis, insulin resistance and severe insulin resistance, diabetic retinopathy, and pancreatitis.

Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples:

EXAMPLES

Example 1 : Gene delivery of Manf to beta-cells of the pancreatic islets averts type 1 diabetes development

1. Methods

1.1 Mice

NOD mice were inbred and housed under semibarrier conditions in an animal facility, and fed a standard chow diet. Ten-week-old female NOD mice were used. Allocation to treatment group was made randomly at weaning, at the cage level. All experiments were performed in accordance with the University of Leuven Animal Ethics Committee guidelines. Sample sizes for mouse experiments were chosen in conjunction with the Animal Ethics Committee to allow for robust sensitivity without excessive use.

1 .2 Diabetes incidence study

Mice were kept until 30 weeks of age and tested twice per week for glucose dysregulation by blood glucose and urine assessment with Diastix Reagent Strips (Bayer). Mice were diagnosed as diabetic when blood glucose went over 250mg/dL (13.9 mmol/L) for two consecutive readings, coupled with a positive urine glucose test. Glucose testing was performed on a blinded basis, with mice being coded by number until experimental end.

1 .3 AAV vector production and purification

AAV production was performed by VectorBuilder (Neu-lsenburg, Germany), using the classical tri-transfection method, with subsequent vector titration performed using a qPCR- based methodology (Fripont et al. (2019) J Vis Exp 29, 143; Rincon et al. (2018) Gene Ther 25, 83-92). For AAV8./ns-Manf, the mouse Manf coding sequence (accession number NM_029103.4) was cloned into a single stranded AAV2-derived expression cassette, containing the 705bp rat Insulin 2 promoter, woodchuck hepatitis post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation (bGH polyA) sequence. Control vectors (AAV8./ns-GFP) were prepared by swapping the Manf coding sequence for that encoding enhanced green fluorescent protein (EGFP, Vector Biolabs). Vector (100 pl total volume) was administered to mice via the interperitoneal route at 1x10 ¹⁰ vector genomes/dose. 2. Results

2.1 Gene delivery of Manf prevents diabetes development in vivo

The identified association between defective Manf production and beta-cell fragility suggested that excess Manf production could be protective in the context of diabetes (Dooley et al. (2016), supra). The present inventors developed a gene delivery-based therapeutic system to deliver Manf to the islets in vivo. An AAV gene delivery system was used to drive the endogenous production of Manf in the islets, using the AAV8 capsid and the rat insulin promoter (AAV8./ns-Manf). Pre-diabetic NOD mice, at 10 weeks of age, were treated with either AAV8./ns-Manf or the control AAV8./ns-GFP vector, and monitored for diabetes development (Figure 1). AAV8./ns-Manf treatment in NOD mice reduced the diabetes rate from 58% to 15% (p=0.0277). These results suggest that gene delivery of Manf to the betacells is capable of increasing islet robustness, providing a potential pathway to therapeutic use in diabetes patients. The ability of AAV8./ns-Manf treatment to drive expression of Manf has been demonstrated in Singh etal. (2022) Biomolecules, 12, 1493 (doi: htlps://dpLprg/10.3390/ biom12101493) in which a substantial upregulation of Manf, specifically within the beta-cell compartment was observed, thus indicating that the AAV8./ns-Manf vector is capable of efficient beta-cell specific gene delivery of Manf.

3. Discussion

The Glis3-Manf pathway appears to be an important fulcrum for diabetes development. There are several lines of evidence indicating that GLIS3 is also a key anti-apoptotic mediator in humans. Using in vitro systems, exposure of human islets to certain dietary fats, such as palmitate and oleate, triggers apoptosis of beta-cells (Laybutt et al. (2007) Diabetologia 50, 752-763; Lupi et al. (2002) Diabetes 51 , 1437-1442; El-Assaad et al. (2003) Endocrinology 144, 4154-4163; Cunha et al. (2016) Cell Death Differ 23, 1995-2006). This effect is accompanied by a reduction in GLIS3 expression (Hall et al. (2014) BMC Med 12, 103; Cnop et al. (2014) Diabetes 63, 1978-1993), and beta-cell apoptosis (in response to palmitate or inflammatory cytokines) is compounded by GLIS3 knockdown (Nogueira et al. (2013), supra). GLIS3 polymorphism being linked to susceptibility to both T1 D and T2D (Barrett et al. (2009) Nature genetics 41 , 703-707; Dupuis etal. (2010) Nature genetics 42, 105-116; Li etal. (2013) Diabetes 62, 291-298; Cho et al. (2011) Nature genetics 44, 67-72), and rare mutations also causing neonatal diabetes (Senee et al. (2006) Nature genetics 38, 682-687), demonstrating that expression variation can modify diabetes risk. In mouse models of beta-cell stress, decreased expression of Glis3 (from heterozygous status, or downstream of high fat diet exposure) sensitised to beta-cell death following islet stress (Dooley et al. (2016), supra).

Manf is a critical survival factor for pancreatic beta cells, with Manf-deficient mice developing spontaneous diabetes due to beta cell apoptosis (Lindahl et al. (2014), supra). Manf demonstrates one of the largest increases in expression following induction of the unfolded protein stress response (Dooley et al. (2016), supra), suggesting it is a programmed stressresponse pathway that enables continued survival. The same process is conserved in humans, with the addition of recombinant MANF protecting human pancreatic beta-cells from stress-induced apoptosis (Hakonen et al. (2018) Diabetologia 61 , 2202-2214). Evidence suggests that effective MANF upregulation during stress requires Glis3 expression. When Glis3 expression is impeded, either through genetic deficiency or diet-induced deficiency, Manf upregulation in response to stress is stunted (Dooley et al. (2016), supra). Likewise in human T2D islets, a positive relationship is observed between GLIS3 expression levels and MANF expression levels (Dooley et al. (2016), supra). Together, this suggests that the anti- apoptotic effect of GLIS3 in human beta-cells may be mediated by MANF.

The use of an AAV-based gene delivery system raises the potential for translation to the human context. While viral vector-based therapeutics have had delayed uptake, improved safety profiles of modern vectors are driving a renaissance in gene delivery and gene therapy clinical trials, with nearly half of the currently open clinical trials based on AAVs. An improved robustness of beta-cells during cellular stress could be clinically beneficial in three different clinical contexts. First, and analogous to the NOD system used herein, the system could be used to protect against T1 D. Improved genetic and serology-based prediction may allow the identification of children pre-disposed to T1 D, for treatment prior to clinical onset. Alternatively, recent-onset T1 D patients may be treated. The “honeymoon phase” that recent-onset T1 D patients only enter following exogenous insulin treatment suggests that both remaining betacell mass is present, and also that the retained cells are operating in a sub-optimal manner due to excessive metabolic stress from insulin production. Increased robustness of these islets may prolong the honeymoon phase and reduce dependence on exogenous insulin. Second, the system could be used to improve islet transplantation. Transplanted islets have poor survival rates, and the transplantation process could be used as a window for exposure to AAV-based MANF buffering. Highly robust transplanted islets have the potential to increase long-term insulin production in patients. Third, the system could have utility in T2D patients. While initial stages of T2D are characterised by insulin-resistance, approx. 30% of patients progress to insulin-dependency, with beta-cell mass being reduced due to chronic metabolic stress. This insulin-dependent T2D stage is refractory to treatment, with a need for exogenous insulin coupled to insulin resistance, leading to high rates of secondary pathologies, such as diabetic foot or diabetic retinopathy. Treatment of early T2D patients at risk of progressing to insulin-dependency therefore has the potential for high clinical impact in an at-risk population.