Patent Application
20140045923
The present invention relates to gene therapy compositions that comprise vectors with at least one sequence that encodes insulin-like growth factor 1 (IGF-I), which will be used to prevent or treat autoimmune diseases. Moreover, the invention comprises the method of preventing or treating type 1 diabetes (DT1) by the expression of IGF-I, specifically in hepatic cells. IGF-I was capable of stopping the immune attack against pancreatic β cells, thereby preventing the loss of β cell mass and, consequently, maintaining circulating insulin at normal levels.
Therefore, the present invention belongs to the field of gene therapy, in particular the gene therapy that focuses on the prevention or treatment of autoimmune diseases.
DT1 is the result of the autoimmune destruction of pancreatic β cells, which leads to insulin deficiency and hyperglycemia (Eizirik et al., 2009). The onset of spontaneous DT1 in human beings is preceded by a progressive leukocyte infiltration in the islets (insulitis), which persists for a relatively long period of time prior to the massive destruction of islet β cells (Atkinson and Eisenbarth, 2001). DT1 is diagnosed when the destruction of β cells is almost complete (Couper et al., 1991) and patients need insulin replacement therapy to survive. Therefore, preventive interventions aimed at interrupting the immune attack against the islets and preventing the loss of β cells are of great interest.
Several growth factors have shown protective properties against DT1 in different animal models. Amongst them, IGF-I is an interesting candidate for an anti-diabetes gene for several reasons. In the first place, it has anti-apoptotic effects in vitro in islets exposed to proinflammatory cytokines (Giannoukakis et al., 2000). In the second place, the daily subcutaneous administration of recombinant human IGF-1 to pre-diabetic NOD (non-obese diabetic) mice reduces the severity of insulitis and the incidence of DT1, which demonstrates the efficacy of the protection of IGF-I against DT1. However, there is a marked adverse effect minutes after the administration of free IGF-I: the circulating levels of glucose decrease, causing lethal hypoglycemia in some animals. This adverse effect is due to the insulin-like hypoglycemic action of IGF-I. Hypoglycemia and its potentially lethal resulting adverse effects, jointly with its impact on the neurocognitive function and emotional well-being, continue to represent a serious danger and a very significant obstacle for achieving a close glycemic control. Moreover, following the subcutaneous administration of recombinant IGF-I, the onset of diabetes is only delayed from day 16 to day 18, but the final incidence of the disease beyond day 22 after the induction of diabetes is probably the same for untreated and treated groups (Bergerot et al., 1995).
In particular, Thivolet et al., in U.S. Pat. No. 6,342,227, disclosed the administration of IGF-I as a method of delaying the clinical onset of diabetes. In their study, Thivolet et al. administered the recombinant IGF-I twice a day, probably because the half-life of IGF-I in the circulation is 9 min, although it can be further increased to 16 hours when it is bound to insulin-like growth factor binding proteins (IGFBP) (Janssen, 2008). In Thivolet's study, it was proposed that the protective action of IGF-I would arise due to the changes in the transit of compromised self-reactive T cells towards the islets of the recipient mouse. I.e. following the transfer of self-reactive T cells from diabetic donours, the treatment with IGF-I reduced the number of donour T cells in the spleen of the recipients.
In previous works at our laboratory, we demonstrated that the local expression of IGF-I in the pancreas of transgenic mice prevents infiltration of the islets and the death of β cells in a DT1 model, the RIP/hIFNβ mouse, which presents an increased susceptibility to developing diabetes (Casellas et al., 2006). Moreover, the expression of IGF-I in β cells of diabetic mice is capable of regenerating the endocrine pancreas in a transgenic model (George et al., 2002).
Taking into consideration the information comprised in the state of the art, the present invention focuses on the discovery of an alternative method capable of preventing or treating DT1, whilst resolving the problems associated with the methods known in the state of the art. In particular, the method developed in the present invention was capable of preventing or treating DT1 by means of a weekly hydrodynamic injection of a plasmid that allowed for the expression of IGF-I in the liver, whilst maintaining normal circulating levels of IGF-I. At no time was any abnormal increase in the levels of circulating free IGF-I observed. Moreover, the number of administrations necessary to prevent or treat DT1 in the present invention could be reduced, under the appropriate conditions, to a single injection. In a clinical environment, a single-dose regimen per treatment, or even a weekly application, would greatly increase the patients' quality of life. The strategy used in the present invention was capable of reducing the incidence of the disease by up to 80% and providing complete protection against DT1 after 10 administrations, even after withdrawing the treatment. Finally, the DNA-based treatment of the invention is more economical, easier to produce and safer than the administration of protein, and does not require refrigeration for short- or long-term storage.
The invention relates to the specific expression, in both hepatocytes and non-parenchymal hepatic cells, of vectors which comprise at least one sequence that encodes IGF-I. Plasmids are particularly preferred vectors. The adverse hypoglycemic effect caused by the direct administration of IGF-I does not take place following the administration of gene therapy compositions that comprise DNA constructs which include a DNA sequence encoding IGF-I or a functional biological equivalent thereof, associated with at least one vector, particularly plasmids, adequate for the expression of IGF-I or the functional biological equivalent thereof in hepatic cells, probably due to the fact that IGF-I is not intensely synthesised by cells. In the present invention, a sequence of experiments were performed in order to assay the preventive effects of IGF-I against DT1 when administered in the form of a hydrodynamically injected plasmid encoding IGF-I.
One of the essential characteristics of the invention is that the gene therapy compositions and the administration thereof are designed for the expression of IGF-I in hepatocytes and non-parenchymal hepatic cells. This leads to physiological levels of circulating IGF-I, which prevents the undesirable effects that take place when the serum levels of IGF-I increase and cause undesirable effects in other target tissues: as an example, hypertrophia in the cardiac muscle tissue.
Therefore, a gene therapy focus was developed wherein the hepatic administration of a plasmid that expresses IGF-I leads to the prevention of the development of diabetic hyperglycemia in a mouse model of autoimmune diabetes. The interruption of the immune attack against pancreatic β cells in animals treated with IGF-I prevents the loss of β cell mass observed in diabetic mice and, consequently, the levels of circulating insulin are maintained. This protection against the disease could be mediated, at least partially, by an increase in the percentage of intrapancreatic regulatory T cells (Treg cells), since an antibody-mediated depletion of this population makes it impossible to prevent the disease mediated by IGF-I.
More specifically, the present invention showed that the expression of IGF-I in the liver, by means of plasmids that have the CAG or CD68 promoters, was capable of mediating the protection against DT1. However, any promoter that may direct the expression of a transgene in parenchymal and non-parenchymal hepatic cells, such as CMV (van Til et al.), PGK (Uesugi et al.) or acetyl-LDL (Bradshaw et al.; Klein et al. 2007), could be potentially used to express transgenes in hepatic cells and, therefore, should be included within the scope of patent protection conferred to the present invention.
In a preferred embodiment of the invention, the plasmids effectively used to administer IGF-I were pCAG IGF-I WPRE (SEQ ID No.: 1) and pCD68 IGF-I (SEQ ID No.: 2).
Moreover, several vectors have proven to be hepatotropic and, therefore, could act as appropriate vectors to express IGF-I in hepatic cells. The present invention demonstrated that the hydrodynamic injection of a plasmid leads to the transfection of most classes of hepatic cells and, therefore, it is likely that the hydrodynamic injection of a viral vector would also do so. Consequently, the present invention suggests that the hydrodynamic injection of a viral vector that expresses IGF-I would be capable of expressing the transgene in hepatic cells and, therefore, protecting against DT1. Below we list examples of said vectors: adenoviral vectors, high-capacity adenoviral vectors (HC-Ad or gutless), AAV (Adeno-associated viruses), Poxviruses, Lentiviral vectors or viral vectors derived from the Herpes simplex virus.
When extrapolated to animals or human beings with greater body weights, the method of administration used in the experiments with mice, the so-called hydrodynamic injection, must be adapted. A description of a method similar to the hydrodynamic injection, adapted to pigs, is disclosed in Aliño et al. For purposes of the present invention, the method of administration of the gene therapy compositions to human beings is a local injection directly into the liver.
An example of a DNA construct comprised in a viral vector may be observed in
For purposes of the present invention, the functional biological equivalent expression of IGF-I may have a sequence that is identical to the sequence that is naturally found in IGF-I, but it may also have modified sequences that provide a function equivalent to that of a vector wherein said sequences are incorporated. In particular, said modifications may include the deletion of ALU repeated sequences of 3′ UTR. This is only one example and it is non-limiting. The experimental confirmation of the invention claimed has been performed in experimental animal models, particularly in mice, and, therefore, the DNA used in the constructs for the gene therapy of DT1 encoded mouse IGF-I. However, the experimental results obtained may be extrapolated to human beings and, therefore, for purposes of the description of the present invention, the term IGF-I is indifferently applied to a polypeptide of mouse or human origin.
In another preferred embodiment, the sequence that encodes IGF-I is a synthetic IGF-I coding sequence. Said synthetic sequence has a nucleotide sequence different from a natural human IGF-I coding sequence. It is preferable for the sequence to make an optimal use of codons; preferably, at least 50%, 70% or 90% of the codons are optimised. Therefore, in preferred embodiments, the synthetic DNA sequence has a sequence identity of at least 80%, 90%, 95% or 99% with respect to the natural human IGF-I sequence. In a particular preferred embodiment, the synthetic DNA sequence has an identity of at least 95%, more preferably an identity of at least 99%, and, more preferably, an identity of 100% with respect to the natural IGF-I sequence.
Moreover, another embodiment of the preceding vector may contain a regulatory system to adjust the expression of the nucleic acid construct or cassette. The expression “regulatory system”, as used in this document, refers to sequences incorporated into the preceding vectors acting in cis or in trans, which regulate any characteristic of the expression of the nucleic acid of interest, as well as gene products acting in trans which are co-expressed in the cell with the vector described above. Regulatory systems may be used for the positive or negative regulation of the expression from the normal levels of expression or existing levels of expression at the time of regulation. The system contributes to the temporal expression pattern of the polynucleotide.
A related aspect of the invention provides a formulation of a pharmaceutical composition for the administration and expression of an IGF-I gene in a cell, preferably a human IGF-I gene. The formulation includes a vector of the preceding aspect jointly with one or more pharmaceutically acceptable components which may, for example, act by stabilising the vector or increasing the transfection efficacy, but may also provide other functions. In a preferred embodiment, the formulation includes the vector in a solution containing polyvinylpyrrolidone (PVP) at between approximately 0.5% and 50%, preferably PVP at approximately 5%. Preferably, PVP has an average molecular weight of approximately 50,000 g/mol. Additional information is disclosed in document PCT US95/17038. However, another example of a formulation includes the vector with a cationic lipid (for example, as described in U.S. Pat. No. 4,897,355, issued on 30 Jan. 1990) and may also include a colipid, such as a neutral colipid.
Moreover, for purposes of the present invention, the expression “therapeutically effective dose” refers to the quantity of active ingredient that increases or decreases the activity with respect to the activity produced in the absence of the therapeutically effective dose. For purposes of the present description of the invention, the term “therapeutically” also includes the prevention of the onset of the disease. Therefore, for purposes of the present invention, the terms prevention or treatment refer to a preventive therapy or treatment which, on the one hand, may prevent the onset of the autoimmune disease and, on the other hand, may stop the progression of an autoimmune disease already ongoing or initiated, blocking the harmful effects of that autoimmune disease in the body.
For any compound, the therapeutically effective dose may be initially estimated in cell culture assays or animal models, habitually mice, rabbits, dogs or pigs. The animal model may also be used to determine the appropriate concentration interval and the administration route. Said information may be subsequently used to determine the useful doses and administration route in human beings.
The therapeutic efficacy and the toxicity, for example, the DE50 (the therapeutically effective dose in 50% of the population) and the DL50 (the lethal dose for 50% of the population) may be determined by conventional pharmaceutical processes in cell cultures or experimentation animals. The ratio between the doses with toxic effects and the therapeutic doses is the therapeutic index, and may be expressed as the ratio DL50/DE50.
Pharmaceutical compositions are preferred which present high therapeutic indices. The data obtained from cell culture assays and animal studies are used to formulate a dosage interval for human use. The dosage contained in said compositions is preferably within an interval of circulating concentrations that includes DE50, with little or no toxicity. The dosage varies within this interval depending on the dosage form used, the patient's sensitivity and the administration route.
The exact dosage will be determined by a skilled professional. The dosage and the administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. The facts that must be taken into consideration include the severity of the pathology, the subject's general health, age, weight and gender, the diet, the time and frequency of administration, the combination or combinations of drugs, sensitivity reactions and the tolerance/response to the therapy. The normal dosage quantities may vary between 0.1 and 100,000 micrograms, to a total dose of approximately 1 g, depending on the administration route. The literature provides guidance regarding particular dosages and administration methods and is generally available to professionals skilled in the art. Specialists in the art will use different formulations for nucleotides than for proteins or the inhibitors thereof. Similarly, the administration of polynucleotides or polypeptides will be specific for particular cells, medical conditions, locations, etc.
In any of the embodiments described above, any of the pharmaceutical compositions of the invention may be administered combined with other appropriate therapeutic agents. The selection of the appropriate agents to be used in a combined therapy may be performed by a person skilled in the art in accordance with conventional pharmaceutical principles. The combination of therapeutic agents may act synergically to achieve the treatment or prevention of the various disorders described above. Using this approach, it may be possible to achieve therapeutic efficacy with lower dosages of each agent, thereby reducing the possibility of adverse secondary effects.
Any of the therapeutic methods described above may be applied to any subject that needs said therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys and, more preferably, human beings.
All the patents and patent applications cited in this description are expressly included in this document as a reference as a whole. The preceding description describes the present invention in general. A more complete understanding may be obtained by referring to the following specific examples, which are provided for illustrative purposes only, and are not intended to limit the scope of the invention.
The plasmids mentioned above, pCAG IGF-I WPRE (SEQ ID No.: 1) and pCD68 IGF-I (SEQ ID No.: 2) were deposited at the Spanish Type Culture Collection (CECT); University of Valencia, Burjasot Campus, Burjasot (Valencia), Spain, with access numbers CECT7560 and CECT7561, respectively, the deposits at the Budapest Treaty Authorities having been made on Sep. 6, 2009.
FIG. 1.—Immunohistochemical analysis of the expression of GFP and Mac-2 in liver sections. Twenty-four hours post-hydrodynamic injection of plasmids CAG, hAAT and CD68, which express GFP, and 1 week following the injection of vector AAV8 CAG GFP WPRE, the staining with specific anti-Mac-2 antibodies in mouse liver sections was examined. GFP+/Mac-2+ double positive cells were detected in mouse livers which had been injected with plasmids CAG-GFP and CD68-GFP (A-C, G-I), but not following the injection of plasmid hAAT-GFP (D-F). Vectors AAV8 lack Kupffer cell tropism, as revealed by the absence of GFP+/Mac-2+ cells in those animals which were injected with AAV8-CAG-GFP (J-L). The white arrows indicate the location of the Kupffer cells. Scale bars: 18 μm (A-C); 22 μm (D-F); 16 μm (G-I); 24 μm (J-L).
FIG. 2.—Analysis of the expression of GFP by means of RT-PCR in separated CD11b+ cells. The animals were injected with saline solution (B) or 20 μg of pCAG GFP WPRE (C). Twenty-four hours post-injection, hepatic CD11b+ cells were separated and the RNA was extracted. A single specific band for GFP was amplified in the group injected with plasmid DNA, which demonstrates that non-parenchymal cells expressed the GFP transgene. Lane (A) shows the molecular weight marker.
FIG. 3.—Analysis by flow cytometry of the expression of GFP in non-parenchymal hepatic cells following the hydrodynamic injection of plasmid DNA. Different hepatic cell populations were analysed by flow cytometry in those animals which were injected with pCAG and AAV8 CAG. The expression of GFP was detected in macrophages—CD11b+ Kupffer cells—(MΦ), CD11c+ dendritic cells (DCs), CD19+B lymphocytes (BCs), and CD4+ and CD8+ T cells. In all the cell populations analysed, the percentage of GFP+ cells in animals which were injected with plasmid CAG was significantly greater than that obtained from animals injected with vector AAV8, which were practically negative for the presence of GFP. A representative experiment of 4 independent experiments is shown. The results are the mean±standard error of the mean (SEM) of the relative number of GFP+ cells within each population. n=4 mice per group. The Y-axis represents the % of GFP+ cells versus the total number of cells.
FIG. 4.—Prevention of type 1 diabetes by the hepatic expression of IGF-I by means of the hydrodynamic injection of plasmid CAG-IGF-I. (A) Tg IFNβ mice were administered 5 consecutive daily doses of STZ (25 mg/kg) at days 1-5. Tg IFNβ mice not treated with STZ were used as control animals (Con). At day 9, the STZ-Con mice were hydrodynamically injected with plasmid pVAX1, which lacks an expression cassette, or 5 μg of a plasmid that encodes IGF-I under the control of the CAG promoter (STZ-IGF-I). The mice were administered a weekly hydrodynamic injection for 10 weeks. Subsequently, the mice's glycemia was followed up for 6 additional weeks. The numbers indicate the number of weeks of duration of the treatment in the murine animal model. At week 2, an analysis of the replication of B cells, measured as the B cell mass, and an analysis of the presence of insulitis in all the study animals were performed. At week 6, a glucose tolerance test was performed. At week 8, the insulinaemia and the serum levels of IGF-I were measured in the study animals and, at week 16, the B cell mass and the insulitis that they presented was once again analysed in all the animals. The upper arrow indicates the follow-up of the glucose levels (glycemia) that the animals showed throughout the entire study. (B) Most of the mice treated with IGF-I did not develop hyperglycemia following the treatment with STZ. On the contrary, the animals treated with a void plasmid (STZ-Con) became highly hyperglycemic during the first few weeks following the administration of STZ. Those animals not treated with STZ (Con) maintained normoglycemia. The values are shown as the mean±SEM. Only the normoglycemic animals in both STZ-IGF-I groups are shown. The end of the re-administration of pIGF-I is indicated by a dotted line. The Y-axis represents the concentration of glycemia expressed in mg/dl and the X-axis represents the time expressed in weeks. (C) In STZ-Con mice, the incidence of diabetes reached 90% (15/17) at week 8, whereas those animals treated with IGF-I showed a clear resistance to the development of hyperglycemia: 83% (19/23) of the animals were normoglycemic in the STZ-IGF-I group treated with 5 μg of plasmid DNA. None of the Con animals (not treated with STZ) became diabetic (0/19). The animals were considered to be diabetic when 2 consecutive blood glucose measurements were greater than 250 mg/dl. The results shown are representative of 3 independent experiments. The end of the re-administration of pIGF-I is indicated by a dotted line. The Y-axis represents the % of diabetic animals and the X-axis represents the time expressed in weeks.
FIG. 5.—Analysis of the lymphocyte infiltration of pancreatic islets following the treatment with IGF-1. (A) Quantification of the pancreatic islets that presented lymphocyte infiltration. The levels of insulitis were determined in control animals (white bars) and animals treated with STZ (black bars) and with STZ-IGF-I (grey bars) after 15 days of treatment. The Y-axis represents the percentage of lymphocyte infiltration in the pancreatic islets of samples from said animals. The degree of infiltration was measured using the following ranking: not infiltrated (0); peri-insulitis (<25%); moderate insulitis (<50%); severe insulitis (>50%). *p<0.05 vs STZ-control. (B) The insulitis was quantified by means of flow cytometry 15 days after the treatment with STZ. The percentage of infiltrating CD4+ and CD8+ T cells was lower in the animals treated with pCAG IGF-I. The results show the mean±SEM of 3 animals per group. *p<0.05 vs Con. # p<0.05 vs STZ-con. The Y-axes of each of the graphs show the percentages of CD11b+, CD4+ and CD8+ cells, respectively. (C) Detection by immunohistochemistry of the insulin present in pancreatic tissue sections of control animals not treated with STZ (Con), control animals treated with STZ (STZ-Con) and animals treated with STZ-IGF-I, at two weeks (1) and at 4 months (2) from the beginning of the treatment. Magnification 100×.
FIG. 6.—Analysis of the apoptosis and replication of β cells, the β cell mass and the circulating levels of insulin and IGF-I. (A) Two weeks after the injection of STZ, the percentage of apoptotic β cells was determined in mice not treated with STZ (Con), in mice treated with STZ (STZ-Con) and in mice treated with STZ which had been administered a plasmid encoding IGF-I (STZ CAG IGF-I). The results are the mean±SEM of four mice per group. (* p<0.05 versus Con. # p<0.05 versus STZ-Con). The Y-axis shows the percentage of apoptotic β cells. (B) An analysis of the replication of β cells was performed in Con, STZ-Con and STZ CAG IGF-I mice 2 weeks after the treatment with STZ. Three pancreatic sections per animal were immunostained against insulin and Ki67, and the frequency of Ki67-positive β cell nuclei was determined. The results are the mean±SEM of four mice per group. (# p<0.05 versus STZ-Con). The Y-axis represents the percentage of Ki67+ β cells. (C) The β cell mass was measured in mice not treated with STZ (Con) and in mice treated with STZ which were injected with void plasmid (STZ-Con) and treated with IGF-I (STZ IGF-I) four months after the administration of STZ. Nine pancreatic sections of each individual mouse were analysed. The results are the mean±SEM of 4 animals per group. (* p<0.05 versus Con). The Y-axis represents the β cell mass as a percentage with respect to the control animals. (D) The insulinaemia was measured by means of an enzyme-linked immunosorbent assay (ELISA) 7 weeks after the first administration of IGF-I. The normoglycemic animals treated with IGF-I maintained normal insulin values, whereas the STZ-Con animals had very low levels of the hormone. The results are expressed as the mean±SEM of 4 animals per group. (* p<0.05 versus Con). The Y-axis represents the insulin concentration expressed as ng/ml. (E) The serum levels of IGF-I at 7 weeks after the first administration of IGF-I, measured by ELISA, did not show any differences between the groups. The results are the mean±SEM of 4 animals per group. The Y-axis represents the serum levels of IGF-I expressed as ng/ml.
FIG. 7.—Cumulative incidence of diabetes following the HTV injection of a plasmid that expresses IGF-I under the control of a hepatocyte-specific promoter (phAAT-IGF-I). (A) The total RNA was extracted from the liver 24 h after the hydrodynamic injection of 5 μg of phAAT IGF-I and retrotranscribed to cDNA. Specific primers were used to detect the expression of IGF-I from the plasmid following the hydrodynamic injection by means of RT-PCR. Lane 1 shows the molecular weight marker. Lane 2 shows the results in control animals injected with saline solution and lane 3 shows the results of the animals injected with phAAt-IGF-I. (B) The cumulative incidence of diabetes reached approximately 90% (13 of 14 animals) in STZ-Con mice at 2 weeks post-treatment with STZ. The animals that expressed IGF-I under the control of a hepatocyte-specific promoter did not show prevention of diabetes (12/12). The animals were considered to be diabetic when 2 consecutive blood glucose measurements were greater than 250 mg/dl. A representative experiment of 3 separate experiments is shown. The suppression of the treatment with IGF-I is indicated by a dotted line. The X-axis represents the time expressed in days and the Y-axis represents the cumulative incidence percentage of diabetes.
FIG. 8.—Cumulative incidence of diabetes following the hydrodynamic injection of a plasmid that expresses IGF-I under the control of a myeloid-cell-specific promoter (pCD68-IGF-I). (A) The total RNA was extracted from the liver 24 h after the hydrodynamic injection of 5 μg of pCD68 IGF-I and retrotranscribed to cDNA. Specific primers were used to detect the expression of IGF-I from the plasmid following the hydrodynamic injection by means of RT-PCR. Lane 1 shows the molecular weight marker. Lane 2 shows the results in the control animals injected with saline solution and lane 3 shows the results of the animals injected with pCD68 IGF-I. (B) The cumulative incidence of diabetes reached 90% at week 6 in STZ-Con mice (9 of 10 mice). The animals that expressed IGF-I under the control of the CD68 promoter were protected against the disease, since only 40% (5/12) of them became diabetic at week 6 post-STZ. None of the mice that were not treated with STZ developed hyperglycemia. The animals were considered to be diabetic when 2 consecutive blood glucose measurements were greater than 250 mg/dl. A representative experiment of 2 separate experiments is shown. The X-axis represents the time expressed in days and the Y-axis represents the cumulative incidence percentage of diabetes.
FIG. 9.—Percentage of regulatory T cells (Treg) following the treatment with pIGF-I and cumulative incidence of diabetes following the depletion of Treg cells. One month after the induction of diabetes, intrahepatic (A), intrapancreatic (B) and intrasplenic (C) lymphocytes were isolated and analyzed by flow cytometry, following the staining with specific antibodies against CD4, CD25 and GITR. The percentage of Treg in the liver and the pancreas of mice treated with IGF-I had increased, whereas the intrasplenic Treg remained unchanged. The values are shown as the mean±SEM. n=4 animals per group. The Y-axis of graphs (A), (B) and (C) represents the percentage of CD25+GITR+ cells versus CD4+ cells. (D) The depletion of CD4+CD25+ Treg cells using anti-CD25 monoclonal antibody resulted in the loss of the protective effects of IGF-I, since 100% of the animals treated with pIGF-I were diabetic at day 30 post-STZ. Over 80% of the animals which had been administered rabbit serum (NRS) as a control, as well as a void plasmid, also developed the disease. Only one-third of the pIGF-I+NRS animals became diabetic. n=5-6 mice per group. The X-axis represents the time expressed in days and the Y-axis represents the cumulative incidence percentage of diabetes.
FIG. 10.—Schematic representation of the constructs that encode the expression of GFP. Plasmids pCAG, phAAT and pCD68 used for the studies of the expression of indicator genes are shown from top to bottom. Below these, vector AAV8-CAG is shown. The CAG promoter is a hybrid promoter composed of the CMV early/intermediate enhancer, the chicken β-actin promoter and the first intron of the human β-globin gene. It is ubiquitously expressed. The hAAT promoter is a hybrid promoter composed of the hepatocyte control region (HCR) enhancer of apolipoprotein E and the human α-antitrypsin promoter. Its expression is limited to hepatocytes. The CD68 protein is a classic macrophage marker. Its expression should be primarily limited to Kupffer cells. The vectors are not to scale.
FIG. 11.—Schematic representation of the constructs that express IGF-I. Plasmids pCAG, phAAT and pCD68 used for the studies on the preventive actions of IGF-I against type 1 diabetes are shown from top to bottom. The vectors are not to scale.
As described above, a gene therapy focus was developed in the present invention wherein the hepatic administration of a plasmid that expresses IGF-I leads to the prevention of the development of diabetic hyperglycemia in an autoimmune diabetes mouse model. The interruption of the immune attack against pancreatic β cells in the animals treated with IGF-I prevents the loss of β cell mass observed in diabetic mice and, therefore, the levels of circulating insulin are maintained.
In the present invention, the synthesis of IGF-I is performed from vectors that comprise an adequate promoter for expression in hepatic cells. The preferred vectors are plasmids that comprise CAG or CD68 as adequate promoters, resulting in a constant supply of IGF-I. Moreover, in the present invention, the preventive use of IGF-I is achieved by administering a weekly hydrodynamic injection of a solution carrying a plasmid that contains the necessary genetic information for the cells to produce IGF-I. In the present invention, a treatment was established which consisted of one weekly administration of DNA, by contrast with the daily injections used by others in the state of the art. The present invention demonstrated that the animals achieved complete protection against DT1 after 10 administrations of a plasmid that encodes IGF-I, even after the treatment was withdrawn. These points acquire special interest when we take into consideration that the prevalence of diabetes is rapidly increasing in developing countries, where repeated treatments with insulin are usually too costly and inaccessible for many patients, leading to a very heavy sanitary, social and economic burden.
One of the most significant aspects of the present invention is that the therapy with IGF-I reduced the incidence of diabetes: 90% of the mice developed diabetes in the STZ-Control group, whereas 80% of the animals in the group treated with IGF-I did not develop the disease. However, a small percentage of the animals treated with IGF-I became diabetic. Therefore, the treatment with IGF-I does not delay the onset of the disease, but decreases the incidence of DT1. Significantly, after a period of weekly treatments that has been estimated at 10 weeks, the animals are completely protected against the disease and do not develop diabetes when the treatment is withdrawn.
The invention, which uses IGF-I, leads to an intervention in the immune system the final result whereof is the interruption of the autoimmune attack against the pancreatic islets by the infiltration of CD4+ and CD8+ T cells, probably through the involvement of regulatory T cells (Treg). This leads to protection against autoimmune diabetes. Moreover, the results of the experiments demonstrate that IGF-I does not prevent the onset of diabetes when the regulatory T cells have been reduced by means of a specific antibody. These observations relate the protective effect achieved by IGF-I with the presence of regulatory T cells. Treg cells are a specialised sub-population of T cells that act by suppressing the activation of the immune system and, therefore, maintain the homeostasis of the immune system and tolerance to autoantigens. Recent advances in the molecular characterisation of this cell population have firmly established its existence and its critical role in the immune system of vertebrates. Interest in regulatory T cells has increased due to signs in experimental mouse models that demonstrate that the immunosuppressive potential of these cells may be therapeutically utilised to treat autoimmune diseases.
Taking into consideration all these results, it is suggested that this focus would be advantageous for all those autoimmune diseases wherein an attack of the immune cells (such as CD4+ and CD8+ T cells) against the target organ is involved in the development of the disorder.
Examples include type 1 diabetes (Kukreja et al., 2002; Lindley et al., 2005), multiple sclerosis (Viglietta et al., 2004), autoimmune vasculitis (Boyer et al., 2004), rheumatoid arthritis (RA) (Cao et al., 2003) and Langerhans cell histiocytosis (LCH) (Senechal et al., 2007), graft-versus-host disease (Cohen and Boyer, 2006), autoimmune proliferative syndrome type II (Kriegel et al., 2004), atherosclerosis (George, 2008), Crohn's disease (Ricciardelli et al., 2008), myasthenia gravis (Meriggioli et al., 2008), psoriasis (Sugiyama et al., 2005), Graves' disease (Pan et al., 2009), myositis (Banica et al., 2009), ulcerative colitis (Holmen et al., 2006) and lupus erythematosus (Kuhn et al., 2009; Valencia et al., 2007), ankylosing spondylitis (Forger et al., 2009), uveitis (Chen et al., 2008), primary biliary cirrhosis (Lan et al., 2006) and autoimmune hepatitis (Vergani and Mieli-Vergana, 2008).
Examples in animal models include experimental autoimmune encephalomyelitis (EAE) (Luth et al., 2008), experimental autoimmune gastritis (EAG) (McHugh, 2005), lupus-erythematosus-like disease (Kuhn et al., 2009), type 1 diabetes (Chen et al., 2005; Setoguchi et al., 2005), ulcerative colitis (Denning et al., 2005), experimental myasthenia gravis (Sheng et al., 2008), experimental autoimmune myositis (Allenbach et al., 2009) and uveitis (Matta et al., 2008).
In sum, the present invention shows a gene therapy focus wherein the hepatic administration of a vector that expresses IGF-I leads to protection against the development of diabetic hyperglycemia or other autoimmune diseases in both human beings and autoimmune diabetes mouse models. The interruption of the immune attack against pancreatic β cells in the animals treated with IGF-I prevents the loss of β cell mass observed in diabetic mice and, therefore, the levels of circulating insulin are maintained. This protection against the disease is considered to be mediated, at least in part, by an increase in the percentage of both intrahepatic and intrapancreatic Treg cells, since an antibody-mediated depletion of this population hinders the prevention of the disease induced by IGF-I.
Therefore, the first embodiment of the present invention relates to a DNA construct that comprises a nucleotide sequence that encodes IGF-I and an adequate promoter to express said IGF-I in hepatic cells. Preferred promoters such as CAG or CD68 are selected. In a preferred embodiment of the invention, the DNA construct consists of plasmid pCAG IGF-I WPRE, characterised by SEQ ID No.: 1, or plasmid pCD68 IGF-I, characterised by SEQ ID No.: 2.
The second embodiment of the invention relates to the use of said DNA construct for the manufacturing of active pharmaceutical compositions for the treatment of DT1 or any other autoimmune disease, particularly, without being limited thereto, autoimmune diseases caused by the dysfunction or de-regulation of Treg cells.
The third embodiment of the invention relates to a pharmaceutical composition that comprises the DNA construct mentioned above and a pharmaceutically acceptable vehicle.
The excipients, vehicle substances and auxiliary substances must be pharmaceutically and pharmacologically tolerable, such that they may be combined with other compounds of the formulation or preparation and do not have any adverse effects on the organism treated. The pharmaceutical compositions or formulations include those that are suitable for oral or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous), although the best administration route depends on the patient's condition. The formulations may be in the form of individual doses. The formulations are prepared in accordance with methods known in the field of pharmacology. The quantities of active substances to be administered may vary depending on the particularities of the therapy.
The fourth embodiment of the invention relates to a method for the treatment or prevention of DT1 or any other autoimmune disease, which comprises the administration of the aforementioned composition to the patient. The method is characterised in that the composition is administered to the patient by hydrodynamic injection once a week, or even once per treatment. The method was capable of reducing the incidence of DT1 by up to 20% and providing complete protection against DT1 following 10 administrations.
The following examples are intended to illustrate the invention, without limiting the scope thereof, and to demonstrate some important phenomena. In the first place, non-parenchymal hepatic cells (NPC) in the liver and parenchymal cells were capable of expressing the transgene following the hydrodynamic tail vein injection process. In the second place, the hydrodynamic injection of a plasmid that encodes IGF-I leads to the prevention of DT1 in an autoimmune diabetes mouse model. In the third place, the administration of IGF-I in the circulation of muscle or hepatic tissues is not sufficient to prevent autoimmune diabetes in the RIP/hIFNβ model.
Transgenic CD1 mice (N10 generation) that express human interferon beta (IFN-β) in β cells were used. All the experiments were performed in male transgenic mice. The transgenic mice were administered an intraperitoneal injection of STZ (25 mg/kg body weight), dissolved in 0.1 mol/l citrate buffer (pH 4.5) immediately prior to the administration, on 5 consecutive days. The mice were fed with a conventional diet ad limitum and kept in a 12-h light-darkness cycle (the lights were turned on at 8:00 am). The animal care and experimentation processes were approved by the Ethics Committee on Animal and Human Experimentation of the Universitat Autònoma of Barcelona.
The tissues were fixed for 12-24 h in formalin, imbibed in paraffin and sectioned. For the immunohistochemical detection, the sections were incubated at 4° C. overnight with specific antibodies for each protein, washed with PBS 3×5 min and incubated with the corresponding secondary antibodies. For the detection of fluorescence, the sections were incubated with fluorochrome-conjugated secondary antibody for 1 h at room temperature and the nuclei were counterstained with Topro or Hoechst. The bright-field detections were developed using the ABC complex (Vector Laboratories, United Kingdom), which uses 3′3′-diaminobenzidine (DAB) as the chromogenic substrate. The sections were counterstained in hematoxylin from Mayer. For the immunohistochemical detection of insulin, glucagon, somatostatin and pancreatic polypeptide proteins, pancreas were fixed for 12-24 h in formalin, imbibed in paraffin and sectioned. The sections were subsequently incubated at 4° C. overnight with guinea pig anti-porcine insulin antibody (Sigma Chemical, St. Louis, Mo.) at a 1:100 dilution, rabbit anti-human glucagon antibody (Signet Labs, Dedham, Mass.) at a 1:4,500 dilution, rabbit anti-human somatostatin antibody (Serotec, Oxford, United Kingdom) at a 1:750 dilution or rabbit anti-human pancreatic polypeptide antibody (ICN Biomedicals) at a 1:2,000 dilution. As secondary antibodies, peroxidase-coupled rabbit anti-guinea-pig IgG (Dako, Glostrup, Denmark) or biotinylated goat anti-rabbit antibody (Pierce, Rockford, Ill.) and ABC complex (Vector Laboratories, Burlingame, Calif.) were used.
The incidence and severity of insulitis was analysed in 3 paraffin sections per pancreas separated by 100-150 μm, stained against insulin using the aforementioned antibody (section 1.3) and counterstained with hematoxylin. The degree of infiltration of mononuclear cells (percentage of insulitis) was measured using the following classifications: not infiltrated, peri-insulitis (mononuclear cells surrounding islets and ducts but without infiltration of the islet architecture), moderate insulitis (mononuclear cells infiltrating <50% of the islet architecture) and severe insulitis (>50% of the islet tissue infiltrated by lymphocytes and/or loss of the islet architecture).
Pancreas were obtained from transgenic mice and the immunohistochemical detection of insulin was performed in three sections (2-3 μm), separated by 200 μm. The surface area (in square micrometres) of each islet and the surface area of each section were determined using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) connected to a video camera with a colour monitor and an image analyser (analySIS 3.0; Soft Imaging System, Lakewood, Colo.). The percentage of β cell surface area in the pancreas was calculated by dividing the surface area of all the insulin-positive cells in a section by the total surface area of this section and multiplying the result by 100. The β cell mass was calculated by multiplying the weight of the pancreas by the percentage of β cell surface area.
The replication of β cells was measured by the immunohistochemical analysis of the Ki67 marker. Ki67 is expressed at low levels during the initial G1 phase and is accumulated during the following S, G2 and M phases of the cell cycle; therefore, it is habitually used as a replication marker.
The samples were deparaffinised and treated with Proteinase K (10 μg/ml) with Tris/HCl, pH 7.6, for approximately 10 min at 37° C., and permeabilised with Triton X (0.1%) in PBS for 2 min. Finally, a double staining was performed using anti-insulin antibody (1:100) and anti-Ki67 antibody (1:200) in order to detect the replication of β cells. These primary antibodies and their secondary antibodies, jointly with the labelled streptavidin and Hoechst (used to counterstain the nuclei and discard false positive cells), are described in Section 3 of this chapter. At least 1000 nuclei of islet β cells per pancreas were counted and four animals per group were examined.
Apoptotic cells in deparaffinised pancreatic sections were identified using transferase-mediated dUTP nick-end labelling (TUNEL) (in situ cell death detection kit; Roche Molecular Biochemicals). No β cells were stained with an antibody cocktail (anti-glucagon, anti-somatostatin and anti-pancreatic polypeptide). The nuclei were counterstained with Hoechst (Sigma Chemical). The β cell was considered to be apoptotic when it was positive for TUNEL and negative for staining with an antibody cocktail. In these sections, the layer of non-β cells showed a red cytosolic staining, whereas the TUNEL-positive cells had green nuclei. At least 1000 nuclei of islet β cells per pancreas were counted and 4 animals per group were examined.
For the analysis of non-parenchymal hepatic cells and splenic cells by flow cytometry, the livers and spleens obtained from control mice were mechanically broken up and treated using the flat portion of a 10-ml syringe piston. The homogenate was subsequently run through 30-μm-pore pre-separation filters (Milteny Biotec), to obtain a suspension of a single cell. The cell suspension was gently coated on a Histopaque-1.083 (Sigma-Aldrich) and centrifuged at 800 g for 20 min. The non-parenchymal cells were collected from the interface. Intrapancreatic lymphocytes were obtained from control mice and treated following enzymatic digestion with collagenase, mechanical breakdown and discontinuous density gradient purification, using a Histopaque-1.083 (Sigma-Aldrich).
The following conjugated mAbs were used for the cell staining: PE-labelled and PE-Cy7-labelled anti-CD11b (M1/70), PE-labelled anti-CD4 (GK 1.5), anti-CD8 (53-6.7), anti-CD19 (6D5), FITC-conjugated anti-F4/80 (BM8), PE-conjugated anti-CD49 (BioLegend), FITC-conjugated anti-CD25, PE-CY7-conjugated anti-GITR (YGITR765), PE-conjugated anti-CD11c (HL3) (BD Pharmingen). Antibodies of the same isotype served as controls. The data were obtained in an FC 500 MPL dual-laser cytometer (Beckman Coulter).
A conventional PCR was used to detect the expression of GFP in Kupffer cells separated by FACS and that of IGF-I from plasmid in the total liver, using GoTaq® DNA Polymerase (Promega, United States). The following primers were used: SEQ ID No.: 3 (GFP Fw primer), SEQ ID No.: 4 (GFP Rv primer), SEQ ID No.: 5 (IGF-I Fw primer) and SEQ ID No.: 6 (IGF-I Rv primer).
A hydrodynamic tail vein injection was performed, as described in Liu et al., 1999. Plasmid DNA was diluted in saline solution in a volume (ml) equal to ˜10% of the mean body weight of the animals (grams), and manually injected into the lateral tail vein in less than 5 seconds. Prior to the injection, the animals were placed under a 250-W infrared heat lamp for a few minutes in order to dilate the blood vessels and facilite observation and simpler access to the tail vein. A plastic immobilisation device (Harvard Apparatus) was used to hold the animal for the injection. No anaesthesia was used, since it is not necessary, provided that an appropriate immobilisation device is used. 9.525-mm (⅜ inches) 26G-calibre hypodermic needles (BD) were used, the largest possible needle calibre that perfectly fits the access vein in order to inject the animals.
Statistical analyses were performed using the GraphPad Prism 4.0 computer programme (GraphPad Software, United States). All the data are shown as the mean±SEM, except as otherwise indicated. When the data followed a normal distribution, a t-test was used for independent samples in order to compare 2 groups. The statistical significance was adjusted to P<0.05.
The hydrodynamic tail vein injection of plasmid DNA is a potent tool for the transfer of genes into the mouse liver. It consists of a tail vein injection of saline solution in 5 seconds which contains plasmid DNA, equivalent to 8%-10% of the body weight. Despite the large percentage of hepatic cells that may be transfected using this method, all the data published thus far described that the transfection of the plasmid supplied was limited to hepatocytes (Budker et al, 2006). However, in their expression analyses, Budker et al. did not use specific antibodies against non-parenchymal cells to unequivocally identify the non-parenchymal cells transduced as such. In order to fully characterise the repertoire of transduced hepatic cells, making use of sensitive techniques, such as a specific immunohistochemical analysis, we subsequently analysed whether other types of hepatic cells could also be transfected following the hydrodynamic injection process. Mice were injected using a number of different constructs that express GFP under the control of ubiquitous or cell-specific promoters, as summarised in Table 1. The following constructs were used: a) a plasmid that encodes GFP under the control of a ubiquitous promoter (pCAG GFP WPRE); b) a plasmid that encodes GFP under the control of a hepatocyte-specific promoter (phAAT GFP WPRE); c) a plasmid that encodes GFP under the control of a myeloid-cell-specific promoter (pCD68 GFP), and d) an AAV8 vector that encodes GFP under the control of a ubiquitous promoter (AAV8 CAG GFP WPRE). The mice were sacrificed 24 hours post-injection of the plasmid or 5 days after the injection of AAV8. The livers were extirpated and hepatic sections were stained in order to detect the presence of GFP in hepatocytes or Kupffer cells (Mac2+ cells).
The maps of each corresponding plasmid are shown in
Since Kupffer cells (KC) eliminate large, insoluble waste fragments by phagocytosis, part of the GFP positivity detected in KCs after the injection of plasmid pCAG GFP WPRE could be the result of the phagocytosis of GFP from hepatocytes. However, following the administration of vector AAV8-CAG-GFP, no GFP+ Kupffer cells were detected (
In addition to liver sinusoidal endothelial cells (LSEC) and Kupffer cells, hepatic sinusoids also house resident dendritic cells and a heterogeneous population of intrahepatic lymphocytes, which contains the subpopulation of pit cells which represent liver-specific natural killer cells (NK). In order to fully characterise the repertoire of hepatic NPCs that could be transfected following the hydrodynamic injection process, an analysis by flow cytometry was performed. Mice were hydrodynamically injected with 20 μg of pCAG-GFP WPRE, sacrificed 24 hours later and non-parenchymal hepatic cells were isolated. A set of animals were also injected with 2.5×1011 viral genomes/animal of AAV8 CAG GFP WPRE and the livers were extirpated at 7 days post-injection. The non-parenchymal cells were stained with specific antibodies for the different types of hepatic cells: CD11b for KCs; CD11b for DCs, CD19 for B lymphocytes; CD4 and CD8 for T lymphocytes. An analysis by flow cytometry was performed to detect the presence of GFP+ cells. Approximately 20% of the Kupffer cells and the intrahepatic B lymphocytes, and between 5% and 10% of the dendritic cells and the T lymphocytes were GFP+ after the hydrodynamic injection of plasmid CAG GFP WPRE (
In order to examine whether the expression of exogenous IGF-I in the liver could prevent DT1, Tg IFNβ mice were treated on a weekly basis with a plasmid that expressed IGF-I under the control of the ubiquitous CAG promoter. The Tg IFNβ mice expressed human β interferon in pancreatic β cells. These mice develop manifest diabetes with lymphocyte infiltration of the islets when they are treated with very low doses of streptozotocin, which does not induce diabetes in control mice (Casellas et al., 2006; Pelegrin et al., 1998). It has previously been demonstrated that the local expression of IGF-I in β cells prevents infiltration of the islets and the death of β cells in these Tg IFNβ mice (Casellas et al., 2006).
Experimental diabetes was induced by the administration of daily injections of 25 mg/kg of streptozotocin (STZ) for 5 consecutive days. Seven days after the first administration of STZ, a plasmid that directed the expression of IGF-I under the control of the ubiquitous CAG promoter was hydrodynamically injected, once a week for 10 weeks. The experimental design is indicated in
Following the injection of the plasmid, the blood glucose levels were measured once a week for 16 weeks. Prior to the treatment with STZ, all the animals were normoglycemic. The animals that did not receive the treatment with STZ (Con) remained normoglycemic. Following the treatment with STZ, the blood glucose levels increased above 500 mg/dl in those animals treated with a non-coding plasmid (STZ-Con), whereas approximately 80% of the mice treated with STZ which received the weekly treatment with pIGF-I remained normoglycemic for the entire duration of the experiment, even after interrupting the treatment with IGF-I at week 10 post-STZ (
When the incidence of diabetes was measured in the mice, it was discovered that 90% of the animals were diabetic at 8 weeks post-STZ in the group treated with non-coding plasmid (STZ-Con group), whereas 83% of the animals did not develop diabetes when they were treated with 5 μg of pCAG IGF-I (
These results indicated that the hepatic expression of IGF-I following the hydrodynamic injection was capable of preventing the development of hyperglycemia in a mouse model of type 1 diabetes.
Insulitis was measured 4 months after the treatment with STZ and the degree of insulitis in the normoglycemic mice treated with IGF-I was similar to that observed in the control mice not treated with STZ (
Lymphocyte infiltration of the islets was also determined by flow cytometry. A relative quantification of the infiltrating lymphocytes (CD4+ T and CD8+ T) and macrophages (CD11b+) was performed 2 weeks after the first injection of STZ. A significant increase in CD4+ and CD8+ T cells was discovered in the pancreas of the STZ-Con mice as compared to the control animals not treated with STZ. In the IGF-I group, the number of infiltrating CD4+ and CD8+ T cells was significantly reduced as compared to the STZ-Con mice (
Therefore, the islets of animals not treated with STZ had a normal insulin staining pattern, both at 15 days and at 4 months (
Four months after the treatment with STZ, the animals treated with IGF-I presented a β cell mass similar to that of animals which had not been injected with STZ, whereas the STZ-Con mice showed a strong reduction, of approximately 90% (
In order to study whether the treatment with IGF-I protected β cells against STZ-induced apoptosis in Tg IFNβ mice, an immunohistochemical analysis was performed to detect non-β cells (with a cocktail of anti-glucagon, anti-somatostatin and anti-pancreatic polypeptide antibodies) and TUNEL was performed to identify apoptotic cells. Two weeks after the treatment with STZ, a 2.5-fold increase was detected in TUNEL-positive β cells in the STZ-Con mice as compared to the animals treated with IGF-I which had been injected with STZ (
Moreover, in order to determine whether the treatment with IGF-I induced replication of β cells in animals treated with STZ, a double immunohistochemical analysis for insulin and the Ki67 replication marker was performed. Two weeks after the injection of STZ, a two-fold increase was observed in β cells undergoing replication in the islets of the mice treated with IGF-I which had been injected with STZ, as compared to the STZ-Con animals (
The increase in β cells undergoing replication following the treatment with IGF-I as compared to the STZ-Con mice suggested that the autoimmunity against β cells was blocked (Nir et al., 2007).
The low levels of insulitis detected in the animals treated with IGF-I suggested that the treatment was causing an interruption of the autoimmune attack against β cells. An immunohistochemical detection of insulin was performed in pancreatic sections of Tg IFNβ mice 4 months after the treatment with STZ in order to determine whether the β cell mass was maintained in the animals treated with IGF-I (
The capacity to maintain normoglycemia following the administration of STZ, the interruption of the progression of insulitis and the maintenance of the β cell mass in the animals treated with IGF-I suggested that their pancreas was functional and capable of producing appropriate blood levels of insulin. In order to confirm this, the serum levels of insulin were measured. The animals treated with the void plasmid (STZ-Con) presented approximately 80% lower serum levels of insulin, whereas the controls not treated with STZ (Con) remained normoinsulinaemic (
In order to examine whether an increase in the circulating levels of IGF-I following an HTV injection of pIGF-I could be responsible for the protection against the development of hyperglycemia observed, the levels of total circulating IGF-I were measured 8 weeks after the induction of diabetes. However, no differences in the circulating levels of IGF-I were detected between the groups (
As described above, the CAG promoter is capable of directing the expression of the transgene in hepatocytes and also in several non-parenchymal hepatic cells following the hydrodynamic injection process. In order to further characterise whether the protection against the development of diabetes following the treatment with IGF-I depended on the specific expression of exogenous IGF-I in hepatocytes, a new plasmid construct was examined which expressed IGF-I under the control of a hepatocyte-specific promoter composed of the hepatocyte control region (HCR) enhancer of apolipoprotein E and the human α-antitrypsin promoter (phAAT-IGF). As previously demonstrated in the study, the hAAT promoter is as potent as the GAC promoter in terms of transcriptional activity.
The expression of IGF-I in the liver of the mice that were injected with the plasmid was determined by means of RT-PCR, using specific primers for exogenous IGF-I. Twenty-four hours after the HTV injection of phAAT IGF-I, the expression of IGF-I from the plasmid could only be detected in the animals which had been injected with pCAG IGF-I WPRE (
The IFNβ mice were treated with daily injections of 25 mg/kg of streptozotocin (STZ) for 5 consecutive days. Following the injection of STZ, a plasmid which directed the expression of IGF-I under the control of the hAAT hepatocyte-specific promoter (STZ-hAAT-IGF-I) was hydrodynamically injected every week for 4 weeks. Mice not treated with STZ and STZ-Con mice were included as controls. Two weeks after the injection of STZ, the cumulative incidence of diabetes was approximately 80% in the STZ-Con mice. The animals that expressed exogenous IGF-I under the control of the hAAT promoter did not show protection against diabetes and the cumulative incidence of the disease was similar to that of the STZ-Con mice (
Since the expression of plasmid IGF-I limited to hepatocytes was incapable of suppressing the onset of diabetes, the effects of the expression of IGF-I in other types of hepatic cells was examined. In the liver, the population of non-parenchymal cells is primarily made up of sinusoidal endothelial cells (LSEC), resident tissue macrophages (Kupffer cells), dendritic cells, astrocytes and liver-associated lymphocytes (Blouin et al., 1977). Only recently has it been demonstrated that many liver functions are dependent on the co-operation between non-parenchymal and parenchymal hepatic cells (Kmiec, 2001). Therefore, in order to examine whether the overexpression of exogenous IGF-I in non-parenchymal hepatic cells could lead to a reduction in hyperglycemia, a plasmid was generated which expressed IGF-I under the control of the murine CD68 promoter. CD68 is a macrophage marker and a type I transmembrane protein that is expressed in the endosomal compartment of all the cells of the mononuclear phagocyte lineage, including monocytes, macrophages, microglia, osteoclasts and, to a lesser extent, immature dendritic cells (Gough et al., 2001). Hepatic astrocytes cells also express CD68 (Visas et al., 2003).
The same experimental design indicated in
These results indicated that the overexpression of IGF-I in non-parenchymal hepatic cells was sufficient to prevent the onset of the disease in approximately 60% of the mice treated with STZ. Therefore, non-parenchymal hepatic cells may be involved in the prevention of DT1 in the Tg IFNβ mouse model of type 1 diabetes.
It has been demonstrated that regulatory T cells control autoimmune physiological processes, such as autoimmune diabetes, and it has been demonstrated that variations in the proportions of Treg in the periphery regulate the evolution towards the disease (Belghith et al., 2003). Therefore, a relative quantification of the percentage of intrahepatic, intrasplenic and intrapancreatic Treg (CD4+CD25+ GITR+ T cells) was performed 1 month after the first injection of STZ—after 4 hydrodynamic injections of pCAG IGF-I and pCD68 IGF-I.
A significant increase, of 60% of Treg, was detected in the liver of the animals treated with both CAG IGF-I and CD68 IGF-I, as compared to the STZ Con mice (
Moreover, an approximately two-fold increase in the levels of intrapancreatic Treg was detected in the animals treated with both CAG IGF-I and CD68 IGF-I, as compared to the STZ Con mice (
These data suggested that the treatment with IGF-I induced a local increase in the percentage of intrahepatic and intrapancreatic Treg that could explain the protection against the development of diabetic hyperglycemia observed.
An increase in the percentage of Treg in the liver of the animals treated with pIGF-I one month after the treatment with STZ (
A depletion of CD25+ cells was performed at days 9 and 12 by means of intraperitoneal injections of rat anti-mouse CD25 monoclonal antibody at a dose of 30 μg/mouse (≈1.5 mg/kg). The control mice received rabbit serum.
The cumulative incidence of diabetes in all the groups is shown in
These results indicated that the depletion of Treg in the autoimmune diabetes Tg IFNβ mouse model could be breaking the peripheral tolerance to β cells, leading to the onset of the disease. This also suggested that Treg cells could be involved in the IGF-I-mediated protection against DT1.
In another set of experiments, an experimental diabetic state was induced in RIP-I/hIFNβ mice by administering daily injections of 25 mg/kg of streptozotocin (STZ) for 5 consecutive days. Following the induction of diabetes, a plasmid was electrotransferred which directs the expression of IGF-I, under the control of the ubiquitous CMV promoter (pCMV IGF-I), to the muscles of transgenic IFNβ mice previously treated with STZ (STZ IGF-I). As controls, untreated mice (Con) and mice which had been injected with STZ (STZ-Con) were used. As expected, forty days after the induction of diabetes, none of the animals (0/6) in the Con group had become diabetic, whereas 80% of the mice in the STZ-Con group (4/5) developed hyperglycemia. All the STZ-IGF-I animals (7/7) became diabetic. The animals that developed hyperglycemia showed evident symptoms of diabetes, such as a severe lymphocyte infiltration in the pancreas (insulitis) and an 80% reduction in the levels of circulating insulin as compared to the control animals. These results indicated that the electrotransfer of a plasmid that encodes IGF-I in the muscles of pre-diabetic transgenic IFNβ mice was not capable of protecting against the disease.
| Number | Date | Country | Kind |
|---|---|---|---|
| 200930442 | Jul 2009 | ES | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/ES2010/070478 | 7/9/2010 | WO | 00 | 8/14/2013 |
