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.

Hematopoietic- and neurologic-expressed sequence 1 (Hn1) depletion in B16.F10 melanoma cells promotes a differentiated phenotype that includes increased melanogenesis and cell cycle arrest.

The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity.

Hematopoietic- and neurologic-expressed sequence 1 expression in the murine GL261 and high-grade human gliomas.

The hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a highly conserved protein that is expressed in developing and regenerating tissues. In this study, Hn1 expression was evaluated in human and murine malignant gliomas. Hn1 mRNA and protein were detected in the murine GL261 glioma cell line and in GL261 brain tumors in vivo.

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.

Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity.

Self-complementary adeno-associated viral (scAAV) vectors bypass the requirement for viral second-strand DNA synthesis, but the packaging capacity of these vectors ( approximately 2.4 kb) is significantly smaller than that of conventional AAV vectors ( approximately 4.8 kb).

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors.

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs.

Treatment of human disease by adeno-associated viral gene transfer.

During the past decade, in vivo administration of viral gene transfer vectors for treatment of numerous human diseases has been brought from bench to bedside in the form of clinical trials, mostly aimed at establishing the safety of the protocol. In preclinical studies in animal models of human disease, adeno-associated viral (AAV) vectors have emerged as a favored gene transfer system for this approach. These vectors are derived from a replication-deficient, non-pathogenic parvovirus with a single-stranded DNA genome.

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.

Recombinant adeno-associated and adenoviral vectors for the transduction of pancreatic and colon carcinoma.

Background: Recombinant adeno-associated viral vectors serotype 2 (rAAV2) can transduce several tissues with high efficiency.

Materials And Methods: The transduction efficiency of rAAV2 in pancreatic and colon cancer was compared to that of recombinant adenovirus (rAd) in vitro and in vivo using green fluorescent protein (GFP).

Results: With the exception of SU.

Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.

Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al.

Altering AAV tropism with mosaic viral capsids.

Over the past decade, AAV-based vectors have emerged as promising candidates for gene therapeutic applications. Despite the broad tropism of the first eight serotypes identified, certain cell types are refractory to transduction with AAV-based vectors. Furthermore, for certain applications the targeting of specific cell types is desirable.

Custom adeno-associated virus capsids: the next generation of recombinant vectors with novel tropism.

Recombinant gene delivery vehicles based on adeno-associated virus (rAAV) have emerged as promising vectors for the correction of genetic and acquired human disease states. These vectors possess many characteristics, including low pathogenicity and immunogenicity, and long-term gene expression after a single administered dose, that make them leading candidates for clinical gene therapy applications. Yet, the broad tissue tropism of the available AAV serotypes remains a disadvantage for the safest, most effective in vivo delivery of transgenes to target tissues.

Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus.

Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF.

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding.

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette.

Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus.

Background: Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines.

Materials And Methods: We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells.

Adenovirus-facilitated nuclear translocation of adeno-associated virus type 2.

We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t(1/2) of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h.