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Development of a liver‐specific Tet‐off AAV8 vector for improved safety of insulin gene therapy for diabetes

Abstract Background Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno‐associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)‐induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long‐acting insulin therapy. Methods We have developed a single AAV8 vector with a Tet‐Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector‐treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet‐Off system (pAAV‐Tetoffbidir‐Alb‐luc) regulating a luciferase reporter gene. We subsequently incorporated a furin‐cleavable codon‐optimised human proinsulin cDNA into pAAV‐Tetoffbidir backbone to form the doxycycline inducible pAAV‐Tetoffbidir‐Alb‐hINSco. Results Using STZ‐induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline. Conclusions The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.

body's basal (non-meal) insulin needs throughout the day and night, supplemented by short-acting insulin to provide the postprandial requirement for higher insulin secretion.
Pancreas and islet transplantation have been demonstrated to restore long-term normoglycaemic homeostasis for up to 5-10 years.
However, donor scarcity, specialised skills and infrastructure, and strict exclusion criteria have restricted the application of such procedures to only a small number of severely diabetic patients. 4,5 Moreover, substitution of daily insulin injections for lifelong immunosuppression incurs significant risks of other adverse side effects.
Other forms of biological β-cell replacement therapies using embryonic, induced pluripotent stem cells (iPSCs) and adult stem cells are still experimental. iPSC-derived insulin producing cells would theoretically be the least immunogenic cell source. However, there is evidence that transplanted autologous iPSC-derived β-cells could still be immunogenic, eliciting both auto-and allogeneic immune responses. 6,7 Bespoke treatment with autologous cell transplantation would also be extraordinarily expensive (possibly in the region of a million dollars for each patient) to cover the costs of expansion and large scale differentiation of stem cells.
Lentiviral-based insulin gene therapy has been reported to be efficacious in the long-term treatment of diabetic rodents. 8,9 However, insertional mutagenesis of the integrated virus is of concern when using this approach and vigorous safety validations are required before it can be considered for feasible clinical application. We and others have recently shown that one-time injection of an adenoassociated virus serotype 8 (AAV8) vector carrying the human insulin gene was sufficient to reduce significantly or normalize non-fasting hyperglycaemia of streptozotocin (STZ)-induced diabetic mice for more than 1 year. 10,11 The use of AAV8 has already been shown to be efficacious for the long term treatment of human haemophilia B without any significant safety issues for moe than 6 years. There is little concern over the danger of over-expression of factor IX in these patients because this has no known clinical impact. Restoring constitutive factor IX production to 1-6% of the physiological blood level is sufficent to reduce or abolish frequent bleeding episodes. 12 By contrast, gene therapy of diabetes requires that insulin secretion should not cause hypoglycaemia from inappropriate temporary or permanent overexpression. In the present study, we have built in a biosafety mechanism in the AAV8-insulin vector by incorporating a Tet-Off switch and tested its ability, in diabetic mice, to turn off insulin expression with doxycycline, as well as reactivate expression upon doxycycline withdrawal. The ability to regulate insulin expression is important in preclinical studies and clinical trials whenever constitutive insulin secretion needs to be reduced to avoid hypoglycaemia.
Vanrell et al. 13 developed a compact single Tet-inducible autoregulated expression system, pTet bidir Alb, that allowed liverspecific regulated gene expression in AAV8 vectors driven by a hybrid Tet-on and albumin promoter. They demonstrated rapid induction of the luciferase reporter as well as interleukin (IL)-12 and interferon-γ with doxycycline to treat liver cancer. The sustained liver-secific expression of IL-12 by the same vector was reported by Gil-Fariña et al. 14 We have modified the AAV8 construct to a TetOff bidir system, by switching the doxycycline-activated rtTA chimera to tTA, aiming to switch off insulin expression with doxycycline. In the Tet-Off system, the tetracycline transcriptional activator (tTA) protein is formed by fusing the tetracycline repressor, TetR, from Escherichia coli to the VP16 transactivator from Herpes simplex virus. In the absence of doxycyline, tTA binds to the seven tandem repeats of TetO, which constitute the tetracycline response element (TRE), and activates the adjacent liver-specific albumin promoter. This results in liver-specific expression of the gene of interest. In the presence of doxycycline, tTA detaches from TetO and inactivates transgene expression. 15 We employed a non-diabetic mouse model with the luciferase reporter gene to confirm the liver-specificity and also the kinetics of the pAAV-TetOff bidir -Alb-luc vector using a live imaging system. These experiments provide information for researchers who may be interested in using this vector to switch off their gene of interest under non-diabetic conditions. Using the pAAV-TetOff bidir -hInsco vector system in STZinduced diabetic mice, we showed that insulin gene expression could be reversibly turned off and on, multiple times, without loss of signal.

| Assembly of AAV vectors
Standard cloning methods were used for the cloning of recombinant AAV vectors. The AAV vectors in the present study were modified from the Tet-on AAV vector pAAV-Tet bidir -Alb-luc. 13 pAAV-Tet bidir -Alb-luc was converted to pAAV-Tetoff bidir -Alb-luc by substituting the Tet-On rtTA transactivator sequence flanked by EcoRI-BamHI restriction sites with the Tet-off tTA-Advanced (tTA-2 S ) transactivator sequence (Takara Bio Inc., Otsu, Japan) with the same restriction sites.

| Packaging, purification and titration of viruses
The AAV8 pseudotyped vector particles used in the present study were packaged with the polyethylenimine triple plasmid transfection into 293 T cells using the transgene plasmid, an adenoviral helper plasmid (HGT1) and a chimeric AAV2 Rep-8Cap packaging plasmid (pAAV2-8) at a ratio of 3:10:3, as described in a previous study. 10 The vectors from transfected cells were harvested and purified using discontinuous iodixanol gradient ultracentrifugation. 16 Vector particles were titrated by a quantitative polymerase chain reaction, as described previously, 17 and the results were used to determine the vector dose for the mice.

| Animal studies
NOD.cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were derived from NOD-SCID parental line and STZ-induced diabetic NSG mice have been used as a model for type 1 diabetes in previous studies. 10,[18][19][20] The NSG breeders were purchased from The Jackson Laboratory were euthanized using carbon dioxide. All efforts were made to minimize suffering. Adminstration of doxycycline (Sigma-Aldrich) was by i.
Blood was drawn from the facial vein and processed to obtain serum for a human C-peptide enzyme-linked immunosorbent assay (ELISA).

| Bioluminescence imaging
Mice were anesthetized with 1-2.5% isoflurane, supplied with 2 L/min oxygen and placed in a dark chamber for light acquisition in an IVIS Spectrum In Vivo Imaging System (Perkin Elmer, Waltham, MA, USA).
No blinding was performed. The data were analyzed using Living Image 4 (Perkin Elmer). The substrate, Xenolight D-luciferin (Perkin Elmer), was dissolved in saline at 30 mg/ml and injected i.p. at 160 mg/kg. The region of interest (ROI) covering the whole animal was defined, and quantification of light emission was performed in photons/second before and after luciferin (substrate) injection. Images were obtained at 1-minute intervals (10-second exposures) for the first 12 to 16 minutes after luciferin administration. The peak signal for each mouse was used as the data point for the experiment.

| C-peptide ELISA
Serum C-peptide was measured using an ELISA kit (EZHCP-20 K) for human C-peptide (EMD Millipore, Billerica, MA, USA) at the indicated times in the respective experiments. The antibody was specific for human C-peptide and did not cross-react with mouse C-peptide.

| Statistical analysis
Statistical analysis were performed using the Prism (GraphPad Software Inc., San Diego, CA, USA). The results were expressed as the mean ± SEM. Comparisons of human C-peptide levels in the serum of treated mice in the absence of doxycycline, induced with doxycyline and after withdrawal of doxycycline, were analyzed. Statistical significance was determined using the Holm-Sidak method, with alpha = 5.000%, without assuming a consistent SD. The transfected cells were induced with 0, 0.5 and 1 μg/ml doxycycline for 24 hours. Luciferase activities were assayed using Nano-Glo® dual-luciferase® reporter (NanoDLR™) assay system (Promega). Readings from each well were normalized to signals from co-transfected controls (pSV40-NlucP). Data are reported as the mean ± SEM from triplicate wells in a single experiment 3 | RESULTS

| In vitro characterization of the pAAV-Tetoff bidir -Alb-luc vector
The Tet-Off vector constructs are represented schematically in Figure 1A. were observed in cells exposed to 1, 10, 100 and 1000 ng/ml of doxycycline ( Figure 2B; see also Supporting information, Table S1). We and then compared with parallel cultures in the absence of doxycycline. It was found that 0.1 ng/ml doxycycline did not attenuate the luciferase activity at 24 hours post doxycycline induction. Luciferase activities of cells exposed to 1 ng/ml decreased to 24.1% of the non-induced signal after induction for 24 hours, and returned to 96.7% of the non-doxycycline-induced control at 24 hours upon doxycycline withdrawal. Cells induced with 10 ng/ml of doxycycline decreased to 4.3% of the non-induced level, and returned to 75.2% of the level of control cells that were not exposed to doxycycline.
The luciferase activity of cells that were exposed to 100 and 1000 ng/ml remained low, with no signicant recovery of activity 24 hours after doxycycline withdrawal. A similar trend of reduction in luciferase activity by different concentrations of doxycycline was observed in transfected rat hepatocytes (see Supporting information, Figure S1). The kinetics of the decrease and increase in luciferase activity by the addition and withdrawal of doxycycline, respectively, were examined over 24 hours. Doxycycline at 1 ng/ml reduced luciferase activity by 50% within approximately 10 hours, and to the basal level within 24 hours. Luciferase activity returned to near maximum activity within 24 hours after the withdrawal of doxycycline ( Figure 3A). Transfected cells that were exposed to 10 ng/ml of doxycycline took approximately 5 hours to reduce luciferase activity by The same number of wells and conditions that were continued with doxycycline exposure had doxycycline withdrawn for a further 24 hour, and luciferase activities were assayed at 72 hours after transfection, using Nano-Glo® dual-luciferase® reporter (NanoDLR™) assay system (Promega). Readings from each well were normalized to signals from co-transfected controls (pSV40-NlucP). Data are reported as the mean ± SEM from triplicate wells in a single experiment Kinetics of the switching off and on of Tet-off system in HepG2 (A) doxycycline at 1 ng/ml. (B) Doxycycline at 10 ng/ml. Cells were exposed to doxycycline for 24 hours, 1 day after transfection. Doxycycline was removed for the subsequent 24 hours, and activities were assayed at 0, 1, 3, 6, 12 and 24 hours. Luciferase activities were assayed using Nano-Glo® dual-luciferase® reporter (NanoDLR™) assay system (Promega). Readings from each well were normalized to signals from co-transfected controls (pSV40-NlucP). Dotted lines depicts the time taken for the luciferase signal to reduce by 50%. Data are reported as the mean ± SEM from triplicate wells in a single experiment.
50% ( Figure 3B). However, more than 24 hours was required to regain the maximum signal after doxycycline withdrawal.

| In vivo imaging of luciferase expression of the AAV8-Tetoff bidir -Alb-luc vector
The liver specificity of the vector was confirmed by real time in vivo imaging. AAV8-Tetoff bidir -Alb-luc vector (1.6 × 10 10 vg/mouse) was injected into the tail vein of mice. The background luciferase activity of non-vector-injected control mice before and after luciferin (substrate) injection is shown in the Supporting information ( Figure S2). The signals are very similar between the two and also to that found in the vectorinjected mice before luciferin was injected. The background relative luciferase activity is in the range of 1-1.5 × 10 5 . The pAAV-Tetoff bidir -Alb-luc vector-injected mice, when injected with luciferin, had relative luciferase activities up to 1 × 10 8 , which is 1000-fold above the background signal.
Luciferase expression was monitored using a live imaging system and found to be confined to the liver ( Figure 4A). Upon a single gavage with 0.4 mg of doxycycline, the signal started to decrease when mea-  Figure S3). Doxycycline doses ≤ 0.8 μg per mouse had no effect with respect to inhibiting the expression of luciferase activity (see Supporting information, Figure S3). The same mice (identified by specific Experiment number followed by mouse ID; e.g. AA6_1, AA11_1) could be repeatedly exposed to doxycycline and showed dose-dependent reductions in luciferase expression (Figure 4; see also Supporting information, Figure S3). Higher doxycycline doses lowered luciferase expression more rapidly and to a greater degree, although more time was required to return to the original expression level.

| In vivo switching off of insulin expression in vector-treated diabetic mice
Diabetic mice were injected with 3-5 × 10 10 Figure 7; see also Supporting information, Figure S7).  Figure S8).

| DISCUSSION
Viral insulin gene therapy has been shown to be effective for treating animals with model type 1 diabetes. [8][9][10]21 Although near euglycaemia could be achieved and maintained for a long period of time with a single optimal dose injection without loss of insulin expression or hypoglycaemia in the diabetic mice, the inadvertent administration of a more-than-optimal dose may result in hypoglycaemia, even when the insulin transgene is driven by glucose responsive promoters. 10,11  Blood glucose levels of mice were measured and blood was drawn at the same time for serum human C-peptide ELISA. Doxycycline feed (20 ppm) was given for 8 days, after which blood glucose and serum human C-peptide levels were determined (the lowest threshold detection level of the C-peptide ELISA was 0.05 ng/ml).

FIGURE 8
Effect of doxycycline on human insulin expression, as measured by human C-peptide. Mice were rendered diabetic with STZ and 2.5 × 10 10 vg/mouse AAV8-Tetoffbidir-Alb-hINSco was injected via the tail vein. When the blood glucose had reverted to near normoglycemia over 1 month post injection, blood was drawn from the mice and serum kept frozen for the human C-peptide asssay. Doxycycline was administered until the mice became hyperglycemia, and blood was drawn and the serum kept frozen. The doxycycline was withdrawn and blood was drawn again when the blood glucose of mice returned to near-normoglycemia. Human C-peptide ELISA was performed. Data are reported as the mean ± SEM (n = 7). **p < 0.005 In such instances, it is absolutely necessary to have a mechanism whereby the insulin gene can be attenuated for a controllable duration or turned off for a prolonged period. We would therefore favour the use of the insulin gene therapy approach to provide a sustained base- The addition of sucrose to the drinking water to lessen the bitterness of doxycycline was not an option in the present study because it would affect the blood glucose readouts of the treated mice. Intraperitoneal injections could also be administered independent of the food or fluid intake to switch off the insulin gene. Body weights were inversely correlated with blood glucose levels, which were regulated by insulin expression from the AAV vector. The increase of body weight was an indication of recovery from diabetes because the mice were no longer losing weight from glucosuria. When insulin expression was stalled by doxycycline, the diabetes condition returned with a corresponding weight loss.
The present study addressed the need for a built-in safety mechanism as an important component of insulin gene therapy of diabetes using developing a Tet-Off system. We have also shown this proof of concept strategy to be effective and robust during multiple cycles of switching off and on without the loss of signal over time. The response time for turning off, although not instantaneous, was complete. Oral doxycycline attains peak plasma concentration in 2-3 hours in humans. 29,30 In the context of switching off insulin transcription, subpeak levels will begin to correct hypoglycemia or may already be sufficient to restore normoglycemia within a short period of time. Oral minocycline appears to peak in serum slightly earlier than doxycycline (2 h versus 2-3 h). This further suggests that more rapidly absorbed analogues could be used in place of doxycycline. If the occurrence of hypoglycaemia needs to be rectified immediately, sweet drinks or boluses of glucagon injections could be given as emergency measures to increase the blood glucose rapidly.
This system would also be applicable if the vector dose has been miscalculated inadvertently or if, for any other reason, there is excessive and dangerous insulin hypersecretion, and the vector could not be retrieved and removed. We could maintain the prolonged switching off of insulin by continuous oral intake of a submicrobial dose of a conventional or slow release form of doxycycline or analogues. The patient would have to resume the insulin injections prior to the gene therapy treatment. In addition to the Tet-Off gene casette, the insulin or Tet-Off insulin cassette could be flanked by loxP sites to enable excision of the insulin transgene by cre-recombinase. This will permanently abolish insulin expression by the AAV vector.
There may be concern about the immune response directed against the Tet regulated system. Vanrell et al., 13