Browsing by Subject "AAV"

Adeno-Associated Virus (AAV) is a gene therapy vector with immense clinical importance. However, its use as a template for homology directed repair has come under greater examination particularly for the generation of site-specific recombined Chimeric antigen receptor or CAR T-cells. This is because traditional CAR T-cells generated from retro- or lentiviral vectors have risks for insertional oncogenesis or exhaustion from tonic signaling due to use of a constitutively active promoter, both problems which AAV directed knockins may overcome. In fact, the use of site-specific knockin CAR T-cells have now entered clinical trials for the treatment of CD19+ blood-borne cancers. While the use of these next-gen AAV generated CARs have excelled for liquid tumors, their use for the treatment of solid tumors has lagged. This is due to poor preclinical modeling for solid-tumor directed CARs which take place in immunocompromised mouse models that do not fully recapitulate the tumor microenvironment known to be problematic for infiltrating lymphocytes. Thus, there is a clear need to evaluate these therapeutics in immunocompetent hosts, however, there exist no known AAV serotype that can effectively target murine T-lymphocytes to generate these site-specific knockins. To ameliorate this problem, we employed a capsid evolution from the AAV6 background to generate a murine T-lymphocyte tropic AAV variant dubbed Ark313. Ark313 is vastly superior to the parent serotype in transducing, gene editing and site-specific knockins in murine T-cells. To characterize how this was happening, we employed a genome wide CRISPR knockout screen in murine primary T-cells to reveal the essential factor for Ark313 transduction to be Qa-2, a non-classical MHC-1b molecule. Due to the restricted tissue expression of the Qa-2 antigen, we injected mice systemically with Ark313 and saw it could transduce up to 25% of spleen resident T-cells including naïve/memory/effector subsets when using a self-complementary transgene. Additionally, Ark313 displayed a liver de-targeted tropism reducing potential off target tissue transduction when employing an ubiquitous promoter. Together we have generated a novel tool for the facile genetic manipulation of murine T-cells both ex and in vivo. We believe Ark313 will be a fundamental reagent to employ when interrogating T-lymphocyte immunotherapeutic questions and for investigating immune basic biology. This work lays the groundwork for the development of human lymphocyte targeting AAVs for generating CARs to combat liquid and solid tumors via systemic dosing.

Glycogen storage disease type Ia is an autosomal recessive disorder caused by a mutation in the glucose-6-phosphatase (G6Pase) catalytic subunit, encoded in humans by G6PC. G6Pase dephosphorylates glucose-6-phosphate (G6P) in the liver to generate glucose that can be shuttled to the bloodstream to maintain normoglycemia. Patients with GSD Ia typically present at 6 months of age with sever hypoglycemia, which is lethal if untreated. The current treatment is a strict dietary regimen in which children must be fed every 2 hours overnight or given nasogastric tube feeding, and adults must consume uncooked cornstarch around the clock to maintain normal blood sugar levels. This treatment maintains survival but fails to prevent other symptoms related to metabolism of the excess G6P, and patients develop hepatic adenomas that may become hepatocellular carcinoma later in life, in addition to progressive renal complications.

To overcome the problems persisting during dietary therapy, the Koeberl lab has sought to develop gene therapy approaches that use adeno-associated virus (AAV) vectors to replace the G6pase activity, restoring normoglycemia and normal metabolic processes. However, the vast majority of AAV-delivered genetic material exists as episomes that do not replicate as cells divide, so the effects of AAV gene therapy on GSD Ia mouse and dog models have proven temporary. We hypothesized that driving integration of therapeutic vector genomes into an affected individual's genome would improve beneficial effects' longevity.

We tested several approaches to accomplish this, and have found positive effects using a zinc finger nuclease (ZFN) that targets the mouse safe harbor ROSA26 locus to induce homologous recombination of the G6PC donor vector into the mouse genome. We were able to see an improvement in mouse survival to 8 months of age, an increase in G6Pase activity at 3 months of age, and a decrease in glycogen accumulation at 3 months of age, when the ZFN vector is administered alongside the G6PC vector, compared with mice that received the G6PC vector alone.

We have also taken an alternative approach to overcoming the long-term complications of the current dietary treatment, which would augment rather than replace the current treatment. We have examined several drugs known to induce autophagy in other disease models or cell culture systems, to determine if we could manipulate autophagic activity in G6PC knockdown hepatocytes or GSD Ia mice. We have found positive results using rapamycin, a well-studied MTOR inhibitor, in mice and cells, and have screened several other drugs as well, finding positive effects for bezafibrate, mifepristone, carbamazepin, and lithium chloride, in terms of lipid reduction (which accumulates as a symptom of GSD Ia) and/or LC3-II enhancement, which is reduced in GSD Ia due to downregulation of autophagy during G6P accumulation.

Patients with inborn errors of metabolism (IEMs) constitute a significant portion of the population despite individual IEMs being classified as rare diseases. However, generally these patients have only very poor therapeutic alternatives (1-3). For metabolic liver disorders these encompass substrate reduction, emergency care and in severe cases, organ transplantation. Therefore, development of new therapeutic approaches is urgently needed but limited by availability of preclinical tools.Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency (1-3). Here, we describe a human liver chimeric transgene-free Il2rg−/−/Rag2−/−/Fah−/−/Aavr−/− (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.