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1.
PLoS One ; 5(11): e13820, 2010 Nov 03.
Article in English | MEDLINE | ID: mdl-21072203

ABSTRACT

BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of ß-amyloid peptide (Aß) of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP). METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aß40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y), whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Endoplasmic Reticulum/metabolism , Lactic Acid/pharmacology , Molecular Chaperones/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases/metabolism , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Humans , Hydrogen-Ion Concentration/drug effects , Immunoprecipitation , Membrane Proteins/metabolism , Microscopy, Fluorescence , Peptide Fragments/metabolism , Protein Binding/drug effects
2.
Hum Gene Ther ; 20(12): 1665-78, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19702438

ABSTRACT

Parvovirus B19 has potential as a gene therapy vector because of its restricted tropism for human erythroid progenitor cells in the bone marrow. B19 binds to the cell surface through P antigen and we identified activated beta(1) integrins as coreceptors for internalization. Because differentiation with phorbol ester induces beta(1) integrin coreceptor activity, but cell differentiation is not desirable in gene transfer to human progenitor cells and one of the downstream effectors of phorbol esters is the small GTPase Rap1, the role of Rap1 in the recruitment of beta(1) integrins on hematopoietic cells was examined. Expression of a constitutively active Rap1 (63E) was sufficient to recruit beta(1) integrin coreceptors in erythroleukemic K562 cells by inducing high-affinity integrin conformation. A crucial role of actin polymerization in Rap1-mediated beta(1) integrin recruitment was documented by complete inhibition of the 63E Rap1 effect with low-dose cytochalasin D and by the ability of a constitutively active mutant of the actin cytoskeleton regulator Rac1 to sensitize K562 cells to the pharmacological activation of endogenous Rap1, using the Rap1 exchange factor-specific 8-pCPT-2'-O-Me-cAMP [8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate]. Interestingly, in primary human erythroid progenitor cells, 8-pCPT-2'-O-Me-cAMP was sufficient to significantly increase B19-mediated gene transfer, suggesting that these cells possess the cytoskeleton organization capacity required for efficient recruitment of beta(1) integrins by brief pharmacological stimulation of Rap1 GTP loading. Because 8-pCPT-2'-O-Me-cAMP has been implicated in enhanced homing of progenitor cells, these results identify a novel tool with which to optimize ex vivo B19-mediated gene transfer and potentially improve homing of transduced cells by Rap1-beta(1) integrin activation with 8-pCPT-2'-O-Me-cAMP.


Subject(s)
Cyclic AMP/analogs & derivatives , Erythroid Precursor Cells/drug effects , Gene Transfer Techniques , Genetic Vectors/metabolism , Guanine Nucleotide Exchange Factors/agonists , Integrin beta1/metabolism , Parvovirus B19, Human/metabolism , Thionucleotides/pharmacology , rap1 GTP-Binding Proteins/agonists , Animals , Cell Line , Cyclic AMP/pharmacology , Cytochalasin D/pharmacology , Erythroid Precursor Cells/metabolism , Genetic Vectors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Mice , Parvovirus B19, Human/genetics , rap1 GTP-Binding Proteins/metabolism
3.
Virology ; 381(2): 194-202, 2008 Nov 25.
Article in English | MEDLINE | ID: mdl-18834608

ABSTRACT

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by approximately 68% and approximately 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.


Subject(s)
Dependovirus/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation, Viral , Genetic Vectors/metabolism , Transgenes/genetics , Tyrosine/metabolism , Capsid/metabolism , Casein Kinase II/metabolism , Cell Nucleus/metabolism , Dependovirus/genetics , HeLa Cells , Humans , Phosphorylation , Protein Transport , Transduction, Genetic , Ubiquitination
4.
Proc Natl Acad Sci U S A ; 105(22): 7827-32, 2008 Jun 03.
Article in English | MEDLINE | ID: mdl-18511559

ABSTRACT

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. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.


Subject(s)
Dependovirus/genetics , Genetic Vectors , Point Mutation , Transduction, Genetic , Tyrosine/genetics , Animals , Capsid/metabolism , Cell Nucleus/metabolism , Genetic Therapy , HeLa Cells , Hepatocytes/metabolism , Humans , Mice , Mice, Inbred C57BL , Phosphorylation , Ubiquitination
5.
Hum Gene Ther ; 19(4): 365-75, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18399730

ABSTRACT

Therapeutic levels of expression of the beta-globin gene have been difficult to achieve with conventional retroviral vectors without the inclusion of DNase I-hypersensitive site (HS2, HS3, and HS4) enhancer elements. We generated recombinant adeno-associated viral (AAV) vectors carrying an antisickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer or the erythroid cell-specific human parvovirus B19 promoter at map unit 6 (B19p6) without any enhancer, and tested their efficacy in a human erythroid cell line (K-562) and in primary murine hematopoietic progenitor cells (c-kit(+)lin()). We report here that (1) self-complementary AAV serotype 2 (scAAV2)-beta-globin vectors containing only the HS2 enhancer are more efficient than single-stranded AAV (ssAAV2)-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (2) scAAV2-beta-globin vectors recombine with scAAV2-HS2+HS3+HS4 vectors after dual-vector transduction, leading to transgene expression; (3) scAAV2-beta-globin as well as scAAV1-beta-globin vectors containing the B19p6 promoter without the HS2 enhancer element are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (4) scAAV2-B19p6-beta-globin vectors in K-562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit(+)lin() cells, yield efficient expression of the beta-globin protein. Thus, the combined use of scAAV vectors and the parvovirus B19 promoter may lead to expression of therapeutic levels the beta-globin gene in human erythroid cells, which has implications in the use of these vectors in gene therapy of beta-thalassemia and sickle cell disease.


Subject(s)
Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , Globins/genetics , Transduction, Genetic , Transgenes/genetics , Animals , Cell Line , Enhancer Elements, Genetic/genetics , Gene Expression , Hematopoietic Stem Cells/metabolism , Humans , Mice , Parvovirus B19, Human , Promoter Regions, Genetic/genetics
6.
Hum Gene Ther ; 19(4): 376-83, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18370591

ABSTRACT

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.


Subject(s)
Bone Marrow Transplantation , Cell Lineage , Dependovirus/genetics , Gene Expression , Genetic Vectors/genetics , Hematopoietic Stem Cells/metabolism , Transgenes/genetics , Animals , Blood Cells/cytology , DNA, Recombinant/genetics , Female , Genetic Engineering , Hematopoietic Stem Cells/virology , Male , Mice , Mice, Inbred C57BL , Models, Animal , Time Factors , Transduction, Genetic
7.
Hum Gene Ther ; 19(3): 267-78, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18303957

ABSTRACT

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.


Subject(s)
DNA, Viral/genetics , Dependovirus/genetics , Genetic Vectors , Hematopoietic Stem Cells/virology , Transduction, Genetic , Virus Integration , Animals , Bleomycin , DNA, Viral/metabolism , Dependovirus/metabolism , Female , Genetic Therapy , Genome, Viral , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Proviruses/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transgenes
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