Lyudmyla G. Glushakova

Development of optimized AAV3 serotype vectors: Mechanism of high-efficiency transduction of human liver cancer cells

Our recent studies have revealed that among the 10 different commonly used adeno-associated virus (AAV) serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface-exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single mutants in liver cancer cells in vitro. In immunodeficient mouse xenograft models, direct intratumoral injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intratumoral and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing proapoptotic genes should prove useful for the potential gene therapy of human liver cancers.

Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts

The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit.

An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1α

We evaluated the feasibility of self-complementary adeno-associated virus (scAAV) vector-mediated knockdown of the pyruvate dehydrogenase complex using small interfering RNAs directed against the E1α subunit gene (PDHA1). AAV serotype 8 was used to stereotaxically deliver scAAV8-si3-PDHA1-Enhanced Green Fluorescent Protein (knockdown) or scAAV8-EGFP (control) vectors into the right striatum and substantia nigra of rats. Rotational asymmetry was employed to quantify abnormal rotation following neurodegeneration in the nigrostriatal system. By 20weeks after surgery, the siRNA-injected rats exhibited higher contralateral rotation during the first 10min following amphetamine administration and lower 90-min total rotations (p≤0.05). Expression of PDC E1α, E1β and E2 subunits in striatum was decreased (p≤0.05) in the siRNA-injected striatum after 14weeks. By week 25, both PDC activity and expression of E1α were lower (p≤0.05) in siRNA-injected striata compared to controls. E1α expression was associated with PDC activity (R(2)=0.48; p=0.006) and modestly associated with counterclockwise rotation (R(2)=0.51;p=0.07). The use of tyrosine-mutant scAAV8 vectors resulted in ~17-fold increase in transduction efficiency of rat striatal neurons in vivo. We conclude that scAAV8-siRNA vector-mediated knockdown of PDC E1α in brain regions typically affected in humans with PDC deficiency results in a reproducible biochemical and clinical phenotype in rats that may be further enhanced with the use of tyrosine-mutant vectors.

Adeno-associated virus-vectored gene therapy for retinal disease

Recombinant adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of retinal degeneration in a variety of animal models that mimic corresponding human diseases. AAV vectors possess a number of features that render them ideally suited for retinal gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. In the sheltered environment of the retina, AAV vectors are able to maintain high levels of transgene expression in the retinal pigmented epithelium (RPE), photoreceptors, or ganglion cells for long periods of time after a single treatment. Each cell type can be specifically targeted by choosing the appropriate combination of AAV serotype, promoter, and intraocular injection site. The focus of this review is on examples of AAV-mediated gene therapy in those animal models of inherited retinal degeneration caused by mutations directly affecting the interacting unit formed by photoreceptors and the RPE. In each case discussed, expression of the therapeutic gene resulted in significant recovery of retinal structure and/or visual function. Because of the key role of the vasculature in maintaining a healthy retina, a summary of AAV gene therapy applications in animal models of retinal neovascular diseases is also included.