Effect of CpG Depletion of Vector Genome on CD8 T Cell Responses in AAV Gene Therapy.

Adeno associated viral (AAV) vectors have emerged as a preferred platform for gene replacement therapy and represent one of the most promising strategies to treat monogenetic disorders such as hemophilia. However, immune responses to gene transfer have hampered human gene therapy in clinical trials. Over the past decade, it has become clear that innate immune recognition provides signals for the induction of antigen-specific responses against vector or transgene product.

Engineering and Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity.

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by selection for liver-targeted AAV variants.

Type I IFN Sensing by cDCs and CD4 T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8 T Cells.

Adeno-associated virus (AAV) vectors are widely used in clinical gene therapy to correct genetic disease by in vivo gene transfer. Although the vectors are useful, in part because of their limited immunogenicity, immune responses directed at vector components have complicated applications in humans. These include, for instance, innate immune sensing of vector components by plasmacytoid dendritic cells (pDCs), which sense the vector DNA genome via Toll-like receptor 9.

Biodistribution of adeno-associated virus type 2 with mutations in the capsid that contribute to heparan sulfate proteoglycan binding.

We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other.

Plasmacytoid and conventional dendritic cells cooperate in crosspriming AAV capsid-specific CD8 T cells.

Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8 T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8 T cells against the input viral capsid antigen.

Evaluation of engineered AAV capsids for hepatic factor IX gene transfer in murine and canine models.

Background: Adeno-associated virus (AAV) gene therapy vectors have shown the best outcomes in human clinical studies for the treatment of genetic diseases such as hemophilia. However, these pivotal investigations have also identified several challenges. For example, high vector doses are often used for hepatic gene transfer, and cytotoxic T lymphocyte responses against viral capsid may occur.

Dynamics of antigen presentation to transgene product-specific CD4 T cells and of Treg induction upon hepatic AAV gene transfer.

The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4 T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into mice followed by adoptive transfer of transgenic OVA-specific T cells.

Potential for cellular stress response to hepatic factor VIII expression from AAV vector.

Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein.

Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice.

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions.

Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors.

Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer.

The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses.

Immune tolerance induction to factor IX through B cell gene transfer: TLR9 signaling delineates between tolerogenic and immunogenic B cells.

A subset of patients with severe hemophilia B, the X-linked bleeding disorder resulting from absence of coagulation factor IX (FIX), develop pathogenic antibody responses during replacement therapy. These inhibitors block standard therapy and are often associated with anaphylactic reactions to FIX. Established clinical immune tolerance induction protocols often fail for FIX inhibitors.

Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B.

Background: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.

Differential type I interferon-dependent transgene silencing of helper-dependent adenoviral vs. adeno-associated viral vectors in vivo.

We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection.

Identification of adeno-associated viral vectors suitable for intestinal gene delivery and modulation of experimental colitis.

Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis.

The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver.

Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice.

High Efficiency Ex Vivo Gene Transfer to Primary Murine B Cells Using Plasmid or Viral Vectors.

Primary autologous B-lymphocytes, following gene transfer and re-implantation, have been successfully utilized to prevent autoimmune disease and adaptive responses to therapeutic proteins in several animal models. However, efficient gene transfer to primary B cells requires use of retroviral vectors, which increase the risk of insertional mutagenesis. Here, we evaluated several alternative gene transfer approaches.

High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity.

AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells.

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1alpha subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation.

Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.

Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors.

Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo.

Heart disease is often the end result of inherited genetic defects, which may potentially be treatable using a gene-transfer approach. Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a realistic method for the treatment of such disorders. Here, we demonstrate and compare the natural affinity of specific AAV serotype capsids for transduction of cardiac tissue.

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene.

Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease.

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands.

A new method for recombinant adeno-associated virus vector delivery to murine diaphragm.

Genetically modified mice are important models for evaluation of potential gene therapies for human diseases. However, their small size often precludes the use of clinically feasible methods for vector delivery, therefore, alternative methods must be used. We have developed a gel-based method for delivery of recombinant adeno-associated virus vectors to the mouse diaphragm, an important target organ for many myopathic diseases.

Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice.

Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII and is currently treated by protein replacement therapies. Within the past decade, gene therapy efforts have come to the forefront of novel therapeutics. In this work, a dual-vector approach was employed in which recombinant adeno-associated viral (rAAV) vectors expressing the heavy and light chains of the murine factor VIII gene were delivered either intramuscularly or intravenously to a mouse model of hemophilia A.