RESUMO
Dendritic cell (DC)-based immunotherapy has recently demonstrated a great potential for clinical applications; however, additional progress in the methods of tumor-specific antigen delivery to DCs is necessary for the further development of anti-tumor vaccines. To this end, a capsid-optimized adeno-associated virus serotype 6 (AAV6-T492V+S663V) vector was developed by site-directed mutagenesis of surface-exposed serine (S) and threonine (T) residues, which have a critical role in intracellular trafficking of AAV vectors. This double-mutant AAV6 vector had â¼ 5-fold greater transduction efficiency in monocyte-derived DCs (moDCs) compared with wild-type (WT)-AAV6 vectors. The increase in the transduction efficiency correlated with the improved nuclear translocation of AAV6-T492V+S663V over that of the WT-AAV6 vector. Additional studies of the CD11c promoter identified critical regulatory elements that fit into the AAV expression cassette and drive EGFP expression in moDCs. Development of a chimeric promoter (chmCD11c) that contains functional modules of CD11c and a Simian virus (SV40) enhancer element dramatically increased the EGFP expression in moDCs. MoDCs transduced by the capsid-optimized AAV6 vector carrying human prostate-specific antigen (hPSA) driven by CBA (AAV6-T492V+S663V-CBA-hPSA) or chmCd11c (AAV6-T492V+S663V-chmCD11c-hPSA) generated specific T-cell clone proliferation and superior cytotoxic T lymphocytes (CTLs) with higher killing capability against human prostate adenocarcinoma cells, LNCaP, compared with WT-AAV6 induced CTLs. Taken together, these studies suggest that optimization of capsid and promoter components of AAV vectors can be a useful approach for efficient targeting of moDCs and may prove to be a promising tool for cancer immunotherapy.
Assuntos
Adenocarcinoma/terapia , Capsídeo/imunologia , Células Dendríticas/imunologia , Dependovirus , Vetores Genéticos , Imunoterapia/métodos , Neoplasias da Próstata/terapia , Transgenes , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologiaRESUMO
BACKGROUND AIMS: Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS: We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS: We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS: These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.
Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Transdução Genética/métodos , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Dependovirus , Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Células K562 , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Increased fetal hemoglobin (HbF) levels reduce the symptoms of sickle cell disease (SCD) and increase the lifespan of patients. Because curative strategies for bone marrow transplantation and gene therapy technologies remain unavailable to a large number of patients, the development of a safe and effective pharmacological therapy that increases HbF offers the greatest potential for disease intervention. Although hydroxyurea increases HbF, a substantial proportion of patients fail to demonstrate an adequate response. Pharmacological inhibitors of DNA methyltransferase (DNMT1) and lysine-specific demethylase 1A (LSD1), 2 epigenome-modifying enzymes associated with the multiprotein corepressor complex recruited to the repressed γ-globin gene, are powerful in vivo inducers of HbF. The hematological side effects of these inhibitors limit feasible clinical exposures. We evaluated whether administering these drugs in combination could reduce the dose and/or time of exposure to any single agent to minimize adverse effects, while achieving additive or synergistic increases in HbF. The DNMT1 inhibitor decitabine (0.5 mg/kg per day) and the LSD1 inhibitor RN-1 (0.25 mg/kg per day) administered in combination 2 days per week produced synergistic increases in F-cells, F-reticulocytes, and γ-globin messenger RNA in healthy baboons. Large increases in HbF and F-cells were observed in healthy, nonanemic, and anemic (phlebotomized) baboons. Combinatorial therapy targeting epigenome-modifying enzymes could thus be a useful strategy for producing larger increases in HbF to modify the clinical course of SCD.
Assuntos
Anemia Falciforme , gama-Globinas , Humanos , Animais , Decitabina/farmacologia , Decitabina/uso terapêutico , gama-Globinas/genética , Epigenoma , Hemoglobina Fetal/genética , Anemia Falciforme/genética , Papio/genética , Histona Desmetilases/genética , Histona Desmetilases/uso terapêuticoRESUMO
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.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Globinas/genética , Transdução Genética , Transgenes/genética , Animais , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Parvovirus B19 Humano , Regiões Promotoras Genéticas/genéticaRESUMO
STUDY OBJECTIVE: To review five cases of severe adverse reactions after vaccination with the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in pediatric and adolescent patients with sickle cell disease (SCD), and to evaluate the prevalence of adverse reactions to PPSV23 in patients with SCD by analyzing data from the Vaccine Adverse Event Reporting System (VAERS). DESIGN: Case series and retrospective analysis of data from the VAERS. DATA SOURCES: Medical records from a tertiary care hospital and the VAERS database. MEASUREMENTS AND MAIN RESULTS: Five cases of severe adverse reactions after vaccination with PPSV23 in pediatric and adolescent patients with SCD (aged 2-22 years) were reviewed. The adverse reactions occurred within 24 hours after immunization, and all five patients required medical attention. Analysis of data from the VAERS found that PPSV23 was the most commonly reported vaccine causing adverse events in patients with SCD, accounting for 62% of all vaccine-induced adverse events. This rate is significantly higher than the rate of adverse events related to PPSV23 in patients with human immunodeficiency virus (HIV) or asthma (62% vs 16%, p<0.0001). The reported number of adverse reactions in pediatric patients (< 18 years old) with SCD was 4 times higher than that reported in adult patients (18-39 years old) with SCD. CONCLUSION: The risk of developing severe adverse reactions to PPSV23 is greater in patients with SCD than in patients with HIV or asthma, and especially in pediatric and adolescent patients with SCD compared with their adult counterparts. Health care professionals should closely monitor for potential adverse events after PPSV23 vaccination or revaccination in patients with SCD, adhere to the recommended time interval between PCV13 and PPSV23 administration, and possibly consider avoiding simultaneous administration of other vaccines with PPSV23.
Assuntos
Anemia Falciforme/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/efeitos adversos , Adolescente , Sistemas de Notificação de Reações Adversas a Medicamentos , Anemia Falciforme/complicações , Criança , Pré-Escolar , Humanos , Masculino , Infecções Pneumocócicas/complicações , Estudos Retrospectivos , Adulto JovemRESUMO
Selective drug delivery to hypoxic tumor niches remains a significant therapeutic challenge that calls for new conceptual approaches. Sickle red blood cells (SSRBCs) have shown an ability to target such hypoxic niches and induce tumoricidal effects when used together with exogenous pro-oxidants. Here we determine whether the delivery of a model therapeutic encapsulated in murine SSRBCs can be enhanced by ex vivo photosensitization under conditions that delay autohemolysis to a time that coincides with maximal localization of SSRBCs in a hypoxic tumor. Hyperspectral imaging of 4T1 carcinomas shows oxygen saturation levels <10% in a large fraction (commonly 50% or more) of the tumor. Using video microscopy of dorsal skin window chambers implanted with 4T1 tumors, we demonstrate that allogeneic SSRBCs, but not normal RBCs (nRBCs), selectively accumulate in hypoxic 4T1 tumors between 12 and 24h after systemic administration. We further show that ex vivo photo-oxidation can program SSRBCs to postpone hemolysis/release of a model therapeutic to a point that coincides with their maximum sequestration in hypoxic tumor microvessels. Under these conditions, drug-loaded photosensitized SSRBCs show a 3-4 fold greater drug delivery to tumors compared to non-photosensitized SSRBCs, drug-loaded photosensitized nRBCs, and free drug. These results demonstrate that photo-oxidized SSRBCs, but not photo-oxidized nRBCs, sequester and hemolyze in hypoxic tumors and release substantially more drug than photo-oxidized nRBCs and non-photo-oxidized SSRBCs. Photo-oxidation of drug-loaded SSRBCs thus appears to exploit the unique tumor targeting and carrier properties of SSRBCs to optimize drug delivery to hypoxic tumors. Such programmed and drug-loaded SSRBCs therefore represent a novel and useful tool for augmenting drug delivery to hypoxic solid tumors.
Assuntos
Anemia Falciforme , Sistemas de Liberação de Medicamentos , Eritrócitos , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Eritrócitos/efeitos dos fármacos , Eritrócitos/efeitos da radiação , Feminino , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/farmacocinética , Hemólise , Humanos , Hipóxia , Luz , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Microvasos , Neoplasias/irrigação sanguínea , Oxirredução , Fotólise , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/farmacologia , Baço/metabolismoRESUMO
The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency â¼24-fold over the WT AAV2 vector, and â¼2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.
Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Substituição de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Núcleo Celular/metabolismo , Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Treonina , Transdução Genética , TransgenesRESUMO
Phosphorylation of surface-exposed tyrosine residues negatively impacts the transduction efficiency of recombinant AAV2 vectors. Pre-treatment of cells with specific cellular serine/threonine kinase inhibitors also significantly increased the transduction efficiency of AAV2 vectors. We reasoned that site-directed mutagenesis of surface-exposed serine residues might allow the vectors to evade phosphorylation and thus lead to higher transduction efficiency. Each of the 15 surface-exposed serine (S) residues was substituted with valine (V) residues, and the transduction efficiency of three of these mutants, S458V, S492V and S662V, was increased by up to ≈ 20-fold in different cell types. The S662V mutant was efficient in transducing human monocyte-derived dendritic cells (moDCs), a cell type not readily amenable to transduction by the conventional AAV vectors, and did not induce any phenotypic changes in these cells. Recombinant S662V-AAV2 vectors encoding a truncated human telomerase (hTERT) gene were generated and used to stimulate cytotoxic T cells (CTLs) against target cells. S662V-AAV2-hTERT vector-transduced DCs resulted in rapid, specific T-cell clone proliferation and generation of robust CTLs, which led to specific cell lysis of K562 cells. These studies suggest that high-efficiency transduction of moDCs by serine-modified AAV2 vectors is feasible, which supports the potential utility of these vectors for future human DCs vaccine studies.
Assuntos
Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Células Dendríticas/virologia , Vetores Genéticos , Transdução Genética , Substituição de Aminoácidos , Células Cultivadas , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Serina/genéticaRESUMO
Abstract Our studies have shown that coinjection of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors carrying the enhanced green fluorescent protein (EGFP) gene with self-complementary (sc) AAV2-T cell protein tyrosine phosphatase (TC-PTP) and scAAV2-protein phosphatase-5 (PP5) vectors resulted in an approximately 16-fold increase in EGFP expression in primary murine hepatocytes in vivo [Jayandharan, G.R., Zhong, L., Li, B., Kachniarz, B., and Srivastava, A. (2008). Gene Ther. 15, 1287-1293]. In the present studies, this strategy was further optimized to achieve transgene expression at reduced vector/helper virus doses. These included the use of scAAV helper viruses containing (1) hepatocyte-specific promoters, (2) tyrosine-mutant AAV2 capsids, and (3) additional AAV serotype vectors known to efficiently transduce hepatocytes. The hepatocyte-specific transthyretin (TTR) promoter was approximately 6- to 7-fold more efficient than the Rous sarcoma virus (RSV) promoter; tyrosine-mutant AAV2 capsids were approximately 6- to 11-fold more efficient than the wild-type AAV2 capsids; and the AAV8 serotype helper virus was approximately 16-fold more efficient than AAV2 serotype helper virus. With these modifications, the vector dose of the helper virus could be further reduced by approximately 50-fold. Last, coadministration of scAAV8-PP5 helper virus increased coagulation factor IX expression from an ssAAV2 vector by approximately 7- to 10-fold, thereby achieving therapeutic levels at lower vector doses. No adverse effect on hepatocytes was observed under any of these experimental conditions. The strategy presented here should be adaptable to any ssAAV transgene cassette and, specifically, liver-directed applications of ssAAV2 vectors containing larger genes that cannot be encapsidated in scAAV vectors.