Browsing by Subject "Adeno-Associated Virus"

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 (GSD Ia) is an autosomal recessive metabolicdisorder caused deficiency of glucose-6-phosphatase (G6Pase) resulting from pathogenic variants in the G6PC gene. G6Pase catalyzes the hydrolysis of glucose-6-phosphate to release glucose which can then enter the bloodstream. GSD Ia patients have excess glycogen accumulation mainly in the liver and kidneys and suffer from life threatening hypoglycemia. The current treatment for GSD Ia is dietary therapy that requires patients to frequently consume uncooked cornstarch on a strict schedule. Cornstarch provides a complex carbohydrate that slowly releases glucose to prevent hypoglycemia. This treatment fails to prevent long-term complications associated with GSD Ia including renal failure and the development of hepatocellular adenomas and carcinomas. This lab and others have developed adeno-associated virus (AAV) vector based gene therapies to deliver and therapeutic G6PC transgene to affected tissues in GSD Ia animal models. However, the therapeutic effect is limited as AAV vector genomes are rapidly lost and the biochemical correction declines. Currently no treatment for GSD Ia exists that provides stable, robust expression of G6Pase that can clear glycogen and prevent hypoglycemia. This study employed a novel genome editing approach designed to insert the therapeutic G6PC into the endogenous locus in canine and murine models of GSD Ia. Integration of the transgene into the genome will promote stable expression of G6Pase and prevent the decline of vector genomes and the therapeutic benefit. This genome editing approach utilizes the CRISPR/Cas9 system to generated targeted double stranded DNA breaks at a targeted site in the genome. The G6PC transgene is present in a Donor template with homology to the DNA break to drive homology directed repair (HDR) resulting in the integration of the transgene into the genome. In a canine model of GSD Ia, editing and incorporation of the transgene was achieved in both adult dogs and puppies. Up to 1.0% of alleles were edited in the dog livers and contained the transgene. G6Pase production from the integrated transgene was detected, which correlated with prevention of hypoglycemia during fasting. This demonstrated genome editing in the liver of a large animal model for an inherited metabolic disorder using HDR to insert a therapeutic transgene. A subsequent study in GSD Ia mice also showed incorporation of a G6PC transgene in the mouse genome. Mice were treated with either the Donor transgene vector alone or with both the Donor and a CRISPR/Cas9 vector to assess to role of nuclease activity on integration. Mice treated with both vectors demonstrated improved blood glucose concentrations during fasting, decreased liver glycogen, and increased vector genome copies. Treatment with the pan PPAR agonist bezafibrate increased the efficiency of genome editing. Mice treated with bezafibrate that received both editing vectors had 5.9% of alleles that contained the integrated transgene, whereas only 3.1% of alleles contained the transgene in mice not treated with the drug. This work showed that integration of a therapeutic transgene using CRISPR/Cas9 based genome editing is possible in murine and canine models of GSD Ia. Editing resulted in biochemical correction and sustained transgene expression. These data support the further development of genome editing technologies for GSD Ia and other inherited metabolic disorders.