Gene therapy with neurogenin3, betacellulin and SOCS1 reverses diabetes in NOD mice

Abstract

Islet transplantation for type 1 diabetes is limited by a shortage of donor islets and requirement for immunosuppression. We approached this problem by inducing in vivo islet neogenesis in non-obese diabetic (NOD) diabetic mice, a model of autoimmune diabetes. We demonstrate that gene therapy with helper-dependent adenovirus carrying neurogenin3 (Ngn3), an islet lineage-defining transcription factor, and betacellulin (Btc), an islet growth factor, leads to the induction of periportal insulin-positive cell clusters in the liver, which are rapidly destroyed. To specifically accord protection to these 'neo-islets' from cytokine-mediated destruction, we overexpressed suppressor of cytokine signaling 1 (SOCS1) gene, using a rat insulin promoter in combination with Ngn3 and Btc. With this approach, about half of diabetic mice attained euglycemia sustained for over 4 months, regain glucose tolerance and appropriate glucose-stimulated insulin secretion. Histological analysis revealed periportal islet hormone-expressing 'neo-islets' in treated mouse livers. Despite evidence of persistent 'insulitis' with activated T cells, these 'neo-islets' persist to maintain euglycemia. This therapy does not affect diabetogenicity of splenocytes, as they retain the ability to transfer diabetes. This study thus provides a proof-of-concept for engineering in vivo islet neogenesis with targeted resistance to cytokine-mediated destruction to provide a long-term reversal of diabetes in NOD mice.

Fig. 1. Ngn3-Btc gene therapy leads to only a transient improvement of hyperglycemia in diabetic NOD mice

Non-fasting 9–10AM blood glucose (A) and body weight (B) in diabetic NOD mice are shown. A decrease in blood glucose and maintenance of body weight is seen with Ngn3-Btc treatment, which is lost after 2 weeks after treatment. Values represent mean±SEM. * p≤0.05 as compared to empty virus.

Fig. 2. Ngn3-Btc gene therapy induces insulin-expressing cell clusters in the periportal regions of the liver

(A). Immunohistochemistry for insulin reveals periportal insulin-positive cell clusters only in the diabetic mice treated with Ngn3-Btc. (C) CD3 immunostaining reveals these periportal insulin-positive clusters infiltrated by lymphocytes in the Ngn3-Btc treated mice. A control no primary antibody staining is also shown to demonstrate specificity of the antibody. Higher magnification of the red boxed area in (A) and (C) are shown in panels (B) and (D) respectively. PV – portal vein. Scale bars represent 50 μm (A&C) and 10μm (B&D).

Fig. 3. Ngn3-Btc-RipSOCS1 gene therapy leads to reversal of diabetes in about half of diabetic NOD mice

(A–B) Blood Glucose and body weight display a sustained normalization in 13 of 28 diabetic NOD mice treated with Ngn3-Btc-RipSOCS1 (responders - Ngn3-Btc-RipSOCS1-R). (C) Plasma insulin levels increase significantly only in the responder mice treated with Ngn3-Btc-RipSOCS1; n=3–5. Values represent mean±SEM. * p≤0.05 as compared to controls (SOCS-1 only or Ngn3-Btc).

Fig. 4. Ngn3-Btc-RipSOCS1 gene therapy leads to restoration of glucose tolerance in diabetic NOD mice

(A) Plasma glucose during an IP-GTT, 4 weeks after treatment. The Ngn3-Btc-RipSOCS1 treated responder mice (Ngn3-Btc-RipSOCS1-R) display normal glucose tolerance as compared to the untreated diabetic mice. (B) Plasma insulin during this GTT revealed a significant improvement over untreated diabetic mice indicating in vivo glucose-stimulated insulin secretion. n=3–5. # p≤0.06 as compared to untreated diabetic controls.

Fig. 5. Ngn3-Btc-RipSOCS1 gene therapy leads to islet hormone-positive periportal ‘neo-islets’

(A–E) Immunohistochemistry for insulin (A), proinsulin (C) and combined staining of other islet hormones (B) reveal the presence of hormone-positive cell clusters in the periportal regions of the liver in Ngn3-Btc-RipSOCS1-R mice. The higher magnification of the red boxed area is shown in the inset below each panel in A–C. β-cell transcription factors, Nkx6.1 and Pdx-1 are also seen in the Ngn3-Btc-Rip-SOCS1-R mice (D&E). (F) RT-qPCR for islet hormones and transcription factors from the livers of treated mice 6 weeks after treatment (n=3–5). Values (mean±SEM) are expressed after normalization relative to GAPDH and eEF1γ. PV – portal vein. Scale bars represent 50 μm in all except for the higer magnification insets in A–C wherein the scale bars represent 10μm. * p≤0.05 as compared to controls (non-diabetic and Ngn3-Btc).

Fig. 6. Ngn3-Btc-RipSOCS1 gene therapy leads to islet hormone-positive periportal ‘neo-islets’

Immunohistochemistry for CD3 and TNF-α reveal the presence of activated lymphocytes in the periportal regions of the liver in treated mice (Ngn3-Btc-RipSOCS1 and Ngn3-Btc) but not in control untreated diabetic mice. The higher magnification of the red boxed area in columns 1&3 are shown in columns 2&4 respectively. PV – portal vein. Scale bars represent 50 μm for images in columns 1&3 and 10μm for images in columns 2&4.

Fig. 7. Ngn3-Btc-RipSOCS1 therapy does not alter the diabetogenicity of splenocytes

No difference is observed in the induction of diabetes on adoptive transfer of splenocytes from Ngn3-Btc-RipSOCS1 and RipSOCS1 treated mice into 6 week old female NOD-Scid mice.