Evaluation of Adeno-Associated Viral Vectors for Liver-Directed Gene Transfer in Dogs

This study evaluated six adeno-associated viral (AAV) vectors expressing green fluorescent protein (GFP) from the liver-specific thyroid hormone–binding globulin (TBG) promoter made with novel capsids in canine liver-directed gene transfer. Studies in 1.5-month-old dogs, which were administered vector through a peripheral vein, showed that AAV8 capsid vectors had the most favorable performance profiles. Interestingly, the absolute levels of hepatocyte transduction achieved with AAV8 were lower in dogs compared with what had been achieved in mice and nonhuman primates. Additional studies were performed with AAV8 delivered into the hepatic artery in adult dogs, with higher doses of vector used to assess potential dose-limiting toxicities. These studies showed good transduction on day 7 in one dog that apparently was lost by day 28 in another dog through the generation of GFP-specific T cells. Each adult dog was carefully monitored for any hemodynamic changes associated with vector infusion. Both animals demonstrated mild to moderate hypotension and bradycardia, which appeared to be anesthesia-related, making it difficult to evaluate contributions of the vector

Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways

Percutaneous Transendocardial Delivery of Self-complementary Adeno-associated Virus 6 Achieves Global Cardiac Gene Transfer in Canines

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated-ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6-so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer

Initial assessment of a novel gel column coombs’ test to detect auto- and alloantibodies in dogs

The antiglobulin test detects allo- and autoantibodies on the surface of red blood cells or in plasma and has been used in veterinary medicine to detect immune-mediated hemolytic anemia (IMHA) and hemolytic transfusion reactions. In this study, we introduced a novel canine antiglobulin gel column technique for Direct Antiglobulin Testing (DAT) and Indirect Antiglobulin Testing (IAT). DATs and IATs were performed on 115 dogs from 3 groups: non-anemic, healthy dogs (n=30); dogs with suspect IMHA (n=37) and dogs anemic but without suspect IMHA (n=48). On 28 samples (17 suspect IMHA and 11 non-suspect IMHA) the routine tube technique was also performed. For 60 samples the DAT was performed with both unwashed and washed red blood cells. The gel-based DAT was positive in 30/37 (81%) suspect IMHA samples and 2/48 (4%) non-suspect IMHA samples. All healthy dogs and all test controls were negative. If more stringent criteria for inclusion as suspect IMHA were used 30/33 (91%) were positive. When comparing the gel-based and tube DATs, 15/17 (88%) suspect IMHA were positive via the gel-based test whereas 7/17 (41%) were positive via tube. 11/11 (100%) of the non-suspect IMHA group were negative by both methods. There was 97% correlation between DAT results when the test was performed with unwashed and washed red blood cells. Furthermore, saline and antiglobulin gel columns were used to evaluate blood transfusion compatibility (n=147). 4 dogs with a total of 19 crossmatches had incompatibilities discovered solely via the antiglobulin gel column. One dog had induced anti-DEA 1.1 alloantibodies, one dog had induced anti-Dal alloantibodies and 2 dogs had as yet unidentified alloantibodies. Dal-associated incompatibilities are generally recognized with all crossmatch methods. In conclusion, the canine antiglobulin-containing gel columns appear to be a promising, sensitive and specific laboratory technique which can be used as a simple standardized screening tool in identifying dogs with IMHA (DAT, IAT) as well as in determining alloimmune hemolytic transfusion reactions (antiglobulin enhanced crossmatch)