Vegf-A mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation
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The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment.
Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice.
At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p < 0.05] vs 0.063 ± 0.02 [n = 4] in Gfp mRNA transfected group (GFP) [p < 0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085 ± 0.02 [n = 4] in VEGF vs 0.030 ± 0.004 [n = 4] in no RNA [p < 0.05] vs 0.034 ± 0.01 [n = 5] in GFP [p < 0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048 ± 0.013 [n = 3] in VEGF vs 0.015 ± 0.0051 [n = 4] in no RNA [p < 0.01] vs 0.013 ± 0.0046 [n = 4] in GFP [p < 0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049 ± 0.0042 [n = 8] in VEGF vs 0.029 ± 0.0052 [n = 5] in no RNA [p < 0.05] vs 0.027 ± 0.0056 [n = 4] in GFP [p < 0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292 ± 0.0032 μl [n = 7] in VEGF vs 0.0178 ± 0.0021 μl [n = 5] in no RNA [p < 0.01] vs 0.0129 ± 0.0012 μl [n = 4] in GFP [p < 0.001]).
Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.
KeywordsCell therapy Diabetes Gene delivery Graft revascularisation Islet transplantation Messenger RNA Pancreatic beta cell RNA delivery VEGFA
Gfp mRNA transfected group
In vitro transcription
modified Vegf-A mRNA transfected group
Severe combined immunodeficiency
Vegf-A mRNA transfected group
Vascular endothelial growth factor A
In clinical islet transplantation, it is estimated that up to half of the donor cells are lost in the initial period after transplantation [1, 2, 3, 4]. Excessively high numbers of islet cells, often from multiple donors, are required to restore euglycaemia in type 1 diabetes via intrahepatic islet transplantation [4, 5]. Achieving insulin independence with lower islet masses or by using abundant alternative cell sources are key to making clinical islet transplantation a suitable and widely applicable treatment . The intrahepatic transplantation site currently used in humans has multiple disadvantages that contribute to islet cell loss (reviewed in Staels et al.  and Cantarelli and Piemonti [6, 7]). Alternative transplantation sites are extensively explored for immunological and metabolic benefits or graft-retrieval purposes [8, 9, 10], but their overall benefit is often reduced by delayed graft revascularisation .
The vital role of a functional vascular network makes impaired graft revascularisation one of the main reasons for graft attrition . While pancreatic islets in situ are highly vascularised, revascularisation of transplanted islets takes several weeks and remains suboptimal compared with endogenous islets . Triggering the host’s angiogenic response by providing angiogenic growth factors has been successfully exploited in animal studies to improve graft revascularisation [13, 14]. However, the traditional viral-vector-based methods used in these studies have inherent risks of insertional mutagenesis or vigorous immune reaction . In addition, prolonged overexpression of angiogenic growth factors may negatively influence vessel microarchitecture and islet graft function [16, 17].
In vitro transcribed mRNA represents an emerging class of gene therapy drugs that harbours the potential to circumvent many, if not all, drawbacks of viral-vector-based gene delivery. Its temporary expression provides unique opportunities , as a transient boost in the production and secretion of angiogenic growth factors may suffice to accelerate graft revascularisation. We explored the effect of mRNA-based delivery of the potent angiogenic factor vascular endothelial growth factor A (VEGF-A) in mouse and human islet cells before transplantation.
Cell culture and transfection
Islets were isolated from C57BL/6JRj mice (Janvier, Saint Berthevin, France), cultured overnight, and the peri-islet capsule was digested immediately before mRNA transfection. EndoC-bH3 cells (SARL Endocells, Paris, France) were cultured and immortalising transgenes were excised before transfection as described in Benazra et al. . Human islets were obtained from the Leiden University Medical Center. Human islets isolated at the Leiden University Medical Center that could not be used for clinical transplantation were used in the studies according to national laws and if research consent was available. See electronic supplementary material (ESM) Table 1 for donor characteristics, and ESM Methods.
Mus musculus Vegf-A (also known as Vegfa) was cloned in the vector pEtheRNA. In vitro transcription (IVT) was performed as previously reported . Modified Vegf-A mRNA was produced by incorporating pseudouridine and 5-methyl-cytidine during IVT [21, 22]. The production of mRNA encoding green fluorescent protein (Gfp) was as previously reported . See ESM Methods.
Measurement of insulin secretion
Isolated mouse islets were cultured overnight and transfected the following day as described above. Islets were incubated for 2 h in Ham’s F-10 (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) with either 2 mmol/l or 20 mmol/l glucose. Insulin concentration was measured by ELISA (Mercodia, Uppsala, Sweden). See ESM Methods.
The Ethical Committee for Animal Use of Vrije Universiteit Brussel approved the experiments. 8–12-week-old male C57BL/6JRj mice (Janvier) were used for syngeneic transplantation and 8–12-week-old male severe combined immunodeficiency (SCID)-beige mice (CB17.Cg-PrkdcscidLystbg-J/Crl) (Charles River, Laboratories, L’Arbresle, France) for xenotransplantation of human islets or EndoC-bH3 cells. Mice were housed under standardised conditions (12 h dark/12 h light cycle) and fed a standard diet ad libitum. To evaluate graft vascularisation and size, mice were randomised to a non-transfected, or a Gfp or Vegf-A mRNA transfected group (henceforth referred to as ‘no RNA’, ‘GFP’, and ‘VEGF’) and received either 100 mouse islets, or 1×105 human islet cells or EndoC-bH3 cells under the kidney capsule. To evaluate the metabolic effect of mRNA transfection of the grafts, alloxan-induced diabetic C57BL/6JRj mice were randomised to no RNA, VEGF, or a modified Vegf-A mRNA-transfected group (henceforth referred to as modVEGF), received a marginal mass of 300 syngeneic mouse islets under the kidney capsule , and underwent metabolic measurements. Experimenters were blind to group assignment and outcome assessment. See ESM Methods.
Cells were fixed, embedded and stained for GFP for analysis of transfection efficiency. Islet grafts were fixed and processed as previously reported . Beta cells were stained for insulin and NKX6.1 and alpha cells for glucagon. Biotinylated tomato lectin was used for analysis of in vivo vascularization. See ESM Methods.
The relative mRNA expression levels of Ppia, Rig1, Ifna1 and Mx1 were analysed via qPCR as previously reported . Data are presented as average dCq compared to the reference gene Ppia. See ESM Methods.
One-way ANOVA, two-way ANOVA, Kruskal–Wallis or logrank tests were used as indicated. Data are presented as mean ± SEM. A value of p < 0.05 was considered statistically significant. See ESM Methods.
A pilot experiment with marginal mass islet grafts in alloxan-induced diabetic mice showed no improvement of blood glucose levels in VEGF over no RNA (data not shown). This may be due to an immune reaction to the mRNA-transfected mouse implants engrafted in immunocompetent hosts, as synthetic mRNA can elicit a type 1 interferon response. The expression of the intracellular mRNA receptor retinoic acid-inducible gene 1, Rig1 (also known as Ddx58), and its downstream targets interferon, Ifna1, and myxovirus resistance, Mx1, was indeed strongly increased on mRNA transfection of islets in vitro. Rig1 activation and Ifna1 and Mx1 induction could be efficiently avoided by using the synthetically modified Vegf-A mRNA (see ESM Fig. 3a–c and ESM Results).
modVEGF had enhanced graft revascularisation at day 7 post transplantation to a similar extent as VEGF in the set-up shown in Fig. 1a (data not shown). Next, the effect of mRNA transfection of marginal mass islet grafts on glycemic control in diabetic mice was compared between no RNA, VEGF and modVEGF (the experimental design is shown in ESM Fig. 4a). At 28 days post transplantation, no RNA mice had 2 h fasting blood glucose levels of 16.64 ± 2.85 mmol/l (n = 10), compared with 21.74 ± 3.63 mmol/l (n = 7) in VEGF and 14.02 ± 1.82 mmol/l (n = 12) in modVEGF (see ESM Fig. 4b-d). In the no RNA group, 40% of mice had at least two consecutive blood glucose readings below 14 mmol/l maintained over the metabolic follow-up period of 28 days, compared with 28.57% in VEGF and 58.33% in modVEGF (see ESM Fig. 4e). At day 29 post transplantation, overnight fasting glycaemia was 12.36 ± 2.00 mmol/l (n = 10) in no RNA, 13.07 ± 2.85 mmol/l (n = 7) in VEGF and 9.43 ± 0.61 mmol/l (n = 12) in modVEGF. Mean AUC during IPGTT results at day 29 post transplantation were 2730 ± 176 mmol/l/min (n = 10) in no RNA, 2758 ± 300 mmol/l/min (n = 7) in VEGF and 2457 ± 128 mmol/l/min (n = 12) in modVEGF (see ESM Fig. 4f). Taken together, these results indicate that transplantation of a marginal mass of modified Vegf-A mRNA-transfected mouse islets conferred a relevant, albeit not statistically significant, improvement in metabolic outcome.
The success of engraftment of any cell-based implant critically depends on its rapid integration with the host vasculature . In islet transplantation, the delay in graft revascularisation severely compromises graft survival and function . Experimental delivery of proangiogenic signals has previously been shown as beneficial for islet transplantation . However, the viral-vector-based methods that were used restricted its translational potential. Transfection of IVT mRNA represents an emerging gene therapeutic approach to which the anticipated risks of classic gene therapy do not apply. In the current study, we employed a novel mRNA-based approach to obtain transient VEGF-A overexpression in mouse and human islet cells, thereby preventing the side effects of chronic VEGF-A overexpression, including increased vascular permeability and inflammation [16, 17, 27].
Vegf-A mRNA transfection did not affect islet cell survival or glucose-stimulated insulin secretion in vitro. Vegf-A mRNA transfection immediately before transplantation significantly increased islet graft revascularisation in the early post-transplantation period of both mouse and human islet cell implants. Unmodified mRNA induced a type 1 interferon response in islets, which could be avoided with modified synthetic mRNA to confer mild glucometabolic benefits in vivo.
While our work pioneers the use of mRNA transfection in beta cell transplantation protocols, future studies need to evaluate the potential of (modified) Vegf-A mRNA transfection for emerging alternative beta(-like) cell sources, including those derived from embryonic or induced pluripotent stem cells. The renal subcapsular space allows easy graft retrieval, but being a highly vascularised site, the full benefit of accelerated graft revascularisation may not become apparent [6, 28]. Clinically relevant alternative sites, including the subcutis, that are shown to suffer from limited graft revascularisation should also be tested [6, 7, 28]. More rapid revascularisation will benefit the oxygen and nutrient supply of islet allografts, but could be associated with an earlier influx of inflammatory cells. Combination with effective immunomodulation will therefore remain essential to ensure optimal engraftment. Finally, the success of islet engraftment is determined by factors other than graft revascularisation and immunoprotection, including graft re-innervation . This difficulty places the mRNA strategy in a unique position as it offers a platform by which combinations of therapeutic proteins can be easily delivered to tackle diverse hurdles simultaneously.
Taken together, our mRNA-based approach opens a broad range of experimental opportunities and serves as a proof of principle to improve the success of engraftment of cell-based implants in diabetes and regenerative medicine.
The authors thank V. Laurysens, A. Demarré and E. Quartier (BENE, Vrije Universiteit Brussel, Brussels, Belgium) for technical help. We also thank J. van den Ameele (Andrea Brand Lab, Gurdon Institute, University of Cambridge, Cambridge, UK) for revising the manuscript.
WS, EdK, CG, CH, KT, LB, HH and NDL designed and conceived the experiments. WS, YV, YH, GL and SDG acquired and analysed the data. EdK provided human islets. CH and KT provided Gfp mRNA, Vegf-A mRNA and modified Vegf-A mRNA. WS, YV, YH, CH, KT, CG, LB, HH and NDL interpreted the data. WS and NDL drafted the article. All authors revised the article and approved of the final version. NDL is the guarantor of this work.
The authors acknowledge support by grants from the Research Foundation Flanders (FWO), the VUB Research Council, Stichting Diabetes Onderzoek Nederland, the European Union Sixth and Seventh Framework Program, the Wetenschappelijk Fonds Willy Gepts (WFWG) of the UZ Brussel and the Belgian Federal Science Policy (IAPVII-07). WS is a PhD fellow of Research Foundation Flanders.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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