Interim report on the effective intraperitoneal therapy of insulin-dependent diabetes mellitus in pet dogs using "Neo-Islets," aggregates of adipose stem and pancreatic islet cells (INAD 012-776)

Abstract

We previously reported that allogeneic, intraperitoneally administered "Neo-Islets," composed of cultured pancreatic islet cells co-aggregated with high numbers of immunoprotective and cytoprotective Adipose-derived Stem Cells, reestablished, through omental engraftment, redifferentiation and splenic and omental up-regulation of regulatory T-cells, normoglycemia in autoimmune Type-1 Diabetic Non-Obese Diabetic (NOD) mice without the use of immunosuppressive agents or encapsulation devices. Based on these observations, we are currently testing this Neo-Islet technology in an FDA guided pilot study (INAD 012-776) in insulin-dependent, spontaneously diabetic pet dogs by ultrasound-guided, intraperitoneal administration of 2x10e5 Neo-Islets/kilogram body weight to metabolically controlled (blood glucose, triglycerides, thyroid and adrenal functions) and sedated animals. We report here interim observations on the first 4 canine Neo-Islet-treated, insulin-dependent pet dogs that are now in the early to intermediate-term follow-up phase of the planned 3 year study (> 6 months post treatment). Current results from this translational study indicate that in dogs, Neo-Islets appear to engraft, redifferentiate and physiologically produce insulin, and are rejected by neither auto- nor allo-immune responses, as evidenced by (a) an absent IgG response to the allogeneic cells contained in the administered Neo-Islets, and (b) progressively improved glycemic control that achieves up to a 50% reduction in daily insulin needs paralleled by a statistically significant decrease in serum glucose concentrations. This is accomplished without the use of anti-rejection drugs or encapsulation devices. No adverse or serious adverse events related to the Neo-Islet administration have been observed to date. We conclude that this minimally invasive therapy has significant translational relevance to veterinary and clinical Type 1 diabetes mellitus by achieving complete and at this point partial glycemic control in two species, i.e., diabetic mice and dogs, respectively.

Fig 1. Gene expression profiles of the cNIs administered to study subjects.

(A) All gene expression levels were normalized to those of the P0 IC banks from which the IC component of the cNIs were derived and are given here as Log10 Relative Quantitation (RQ; see Methods). Data are expressed as mean with 95% confidence interval, and all reactions were carried out in duplicate. INS, GCG, SST, PPY, NKX6-1, VEGF, and CXCL12 are all expressed in the cNIs administered to each dog. PDX-1 is no longer expressed. (B) Fold changes in gene expression (mean ± SEM) of the doses given to all dogs combined, versus those of the P0 IC banks from which the IC components were derived. For all treated dogs, their cNI doses express ~4-fold less insulin, ~5-fold less GCG, ~3-fold less SST, ~4-fold less PPY than the P0 ICs from which they were partially derived. Aside from PDX-1, which was not detected in cNI doses, other assessed expression profiles (NKX6-1, VEGF, and CXCL12) were not significantly different from those of the P0 ICs. For both (A) and (B), a difference of ± 2 is considered significant (see Methods).

Fig 2. Serum glucose levels and insulin needs over time.

(A) Serum glucose concentration of study dogs, as assessed and reported by owners, prior to treatment (0 months) and over the study period. As dogs were treated at different times, they are not all currently at the same post-treatment time, thus the reported follow-up period for each is different. (B) Insulin doses for the same time frame. Glucose and insulin dose are reported at leasttwice per day. All values for glucose concentration are averaged for the pretreatment period and for each month post-treatment. Units of insulin administered per day are calculated and averaged for each month. (C) Percent reduction in daily insulin dose at the current follow-up point from baseline and average mg/dL reduction in serum glucose from baseline along with statistical significance (P values) for each dog are shown.

Fig 3. Antigenic responses to cNI treatment, and pre-existing presence of auto-islet antibodies.

AntiASC and anti-IC IgG responses as assessed by FACS in sera of the study dogs before (A) and after (B) treatment with allogeneic cNIs. Shown are percentages of FITC-labeled anti-dog IgG antibody. Sera were collected from dogs before (A) and 1.5 to 3 months after (B) cNI administration. The percent of positive cells is indicated above each column. Dogs do not show increased IgG responses to either ASCs or ICs after cNI administration, indicating there is no additional allo-immune response by the recipients to either cell type. Three dogs show pre-existing antibodies to ICs prior to treatment with cNIs, suggesting they have an autoimmune form of diabetes. (C) Results of ELISA testing of sera from the treated dogs for specific anti-islet antigens, IA2 and GAD65, indicate that none of the dogs have antibodies to GAD65 antigen, but that WSU-01’s serum contained antibodies to IA2 prior to, but not after treatment. Samples were run in duplicate. Note: a pre-treatment serum sample was not available for VSH-01.