Adeno-associated virus-mediated IL-10 gene therapy inhibits diabetes recurrence in syngeneic islet cell transplantation of NOD mice.

Islet transplantation represents a potential cure for type 1 diabetes, yet persistent autoimmune and allogeneic immunities currently limit its clinical efficacy. For alleviating the autoimmune destruction of transplanted islets, newly diagnosed NOD mice were provided a single intramuscular injection of recombinant adeno-associated viral vector encoding murine IL-10 (rAAV-IL-10) 4 weeks before renal capsule delivery of 650 syngeneic islets. A dose-dependent protection of islet grafts was observed. Sixty percent (3 of 5) of NOD mice that received a transduction of a high-dose (4 x 10(9) infectious units) rAAV-IL-10 remained normoglycemic for at least 117 days, whereas diabetes recurred within 17 days in mice that received a low-dose rAAV-IL-10 (4 x 10(8) infectious units; 5 of 5) as well as in all of the control mice (5 of 5 untreated and 4 of 4 rAAV-green fluorescent protein-transduced). Serum IL-10 levels positively correlated with prolonged graft survival and were negatively associated with the intensity of autoimmunity. The mechanism of rAAV-IL-10 protection involved a reduction of lymphocytic infiltration as well as induction of antioxidant enzymes manganese superoxide dismutase and heme oxygenase 1 in islet grafts. These studies support the utility of immunoregulatory cytokine gene therapy delivered by rAAV for preventing autoimmune disease recurrence in transplant-based therapies for type 1 diabetes.

Adeno-associated virus transduction of islets with interleukin-4 results in impaired metabolic function in syngeneic marginal islet mass transplantation.

Previous studies suggest that therapeutic expression of interleukin (IL)-4 by islet cells improves their efficacy in transplantation models directed at reversing type 1 diabetes. We investigated the effects of introducing IL-4 into islets with recombinant adeno-associated virus (rAAV) on the reversal of hyperglycemia in a syngeneic marginal islet mass transplantation model. C57BL/6 islets were mock-transduced or transduced with rAAV expressing murine IL-4 (rAAV-IL-4) or rAAV expressing green fluorescent protein (rAAV-GFP) before transplantation of a marginal mass into diabetic mice. Normoglycemia was achieved in only 1/7 mice receiving rAAV-IL-4 transduced islets in comparison to 6/6 mock-transduced and 4/6 rAAV-GFP transduced animals. The failure of IL-4 expressing islets was not associated with cellular toxicity of rAAV or impairment of glucose-stimulated insulin release in vitro. Islet expression of IL-4 led to impaired metabolic function in mice receiving a marginal mass of syngeneic islets.

Efficient hepatic delivery and expression from a recombinant adeno-associated virus 8 pseudotyped alpha1-antitrypsin vector.

alpha1-Antitrypsin (AAT) deficiency is a single-gene disorder in which a mutation in the AAT (approved symbol SERPINA1) gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs, and a toxic effect on hepatocytes. Optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction to be effective for liver and lung disease. Recently, rAAV genomes pseudotyped with capsids from serotypes 7 and 8 showed efficient hepatic transduction. We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express hAAT in both a greater percentage of cells and greater amounts. AAV2 and pseudotyped vectors for serotypes 1, 5, and 8 carrying the human AAT transgene were injected at 1 x 10(10) particle doses into C57Bl/6 mice. Circulating hAAT from AAV2/8-injected animals showed a 2-log advantage over AAV2 and 3-log increase over AAV2/1 and 5 for the 24-week study. Most significantly, up to 40% of total liver cells stained positive for the transgene in AAV2/8 subjects while remaining primarily episomal. Therefore, pseudotyped AAV8 provides a vehicle to infect a high percentage of hepatocytes stably and thereby express therapeutic molecules to modify AAT PiZ transcripts.

Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects.

We performed a series of studies in baboons to evaluate the safety of intramuscular administration of rAAV vector expressing the alpha-1 antitrypsin (AAT) gene (SERPINA1) in a nonhuman primate model. Initial experiments performed with an rAAV vector expressing the human SERPINA1 gene (at doses of up to 5 x 10(12) vector genomes/kg) resulted in the generation of anti-human AAT antibodies, which correlated with a loss of detectable transgene expression. Subsequent studies made use of the baboon SERPINA1 gene tagged with a short (10-amino-acid) c-myc tag. When animals were sacrificed, 4 months after vector injection, transduced myofibers showed efficient transgene expression without detectable humoral immune responses. Mild inflammation was observed in and near the sites of injection in some vector- and saline-injected animals, but serum creatine kinase (CK) values were normal in nearly every case. Real-time PCR was also performed 4 months after injection on gonadal tissue to evaluate the risk of germline transmission. No vector sequences were detected in the gonadal tissues from these animals. These studies indicate that the risks of immune reaction and germline transmission after intramuscular injection of rAAV-SERPINA1 in nonhuman primates are relatively low within the range of vector doses studied.