RESUMO
The ability to accurately determine the dose of an adeno-associated viral (AAV) therapeutic vector is critical to the gene therapy process. Quantitative PCR (qPCR) is one of the common methods to quantify the AAV vector titre, but different variables can lead to inconsistent results. The aim of this study was to analyze the influence of the conformation of the DNA used as the standard control, and the enzymatic digestion was performed to release the viral genome from the protein capsid on the physical genome titration of a clinically relevant AAV8.RPGR vector, made to good laboratory practice standards in an academic setting. The results of this study showed that the conformation of the DNA used as standard has a significant impact on the accuracy of absolute quantification by qPCR. The use of supercoiled undigested plasmid DNA template generated a higher apparent titer, as compared to the use of linearized plasmid as the standard. In contrast to previous studies, the pre-treatment of the samples with Proteinase K, in addition to the high temperature step used after DNase I digestion, resulted in a reduction on AAV titers. Ideally, all AAV documentation should state which form of reference plasmid and which pre-treatment of the samples have been used to calculate titers, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made in the clinical scenario.
Assuntos
DNA Viral/análise , Dependovirus/genética , Vetores Genéticos/análise , Genoma Viral , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Viral/genética , Vetores Genéticos/genética , Células HEK293 , HumanosRESUMO
As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)
Assuntos
Animais , Bovinos , Zigoto , Animais Geneticamente Modificados/genética , Transgenes , Embrião de Mamíferos , Vetores Genéticos/análise , Fertilização in vitro/veterinária , Técnicas de Transferência de Genes/veterináriaRESUMO
Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time-consuming. Here, we report a method to titer AAV with GelGreen® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (â¼30 min) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid enzyme-linked immunosorbent assay results are comparable with each other. We also showed that GelRed® dye, a red fluorescence dye from Biotium, can be used to directly "visualize" AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. Finally, we showed that GelGreen and GelRed dyes can also be used to quantify self-complementary AAV (scAAV) and crudely purified AAV samples. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable, and cost efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.
Assuntos
DNA Viral/análise , Dependovirus/genética , Corantes Fluorescentes/química , Vetores Genéticos/análise , DNA Viral/genética , Vetores Genéticos/genética , HumanosRESUMO
Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.
Assuntos
Vetores Genéticos/metabolismo , Glicoproteínas/metabolismo , Rede Nervosa/metabolismo , Técnicas de Rastreamento Neuroanatômico/métodos , Vírus da Raiva/metabolismo , Córtex Visual/metabolismo , Animais , Cricetinae , Deleção de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/análise , Vetores Genéticos/genética , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/química , Rede Nervosa/efeitos dos fármacos , Vírus da Raiva/química , Vírus da Raiva/genética , Córtex Visual/química , Córtex Visual/efeitos dos fármacosRESUMO
Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs. Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between naïve and effector cells.
Assuntos
Linfócitos T CD8-Positivos/química , Vetores Genéticos/análise , Spiruroidea/química , Coloração e Rotulagem/métodos , Transgenes , Vacinação/métodos , Animais , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo/métodos , Genes MHC Classe I/imunologia , Vetores Genéticos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/análise , Receptores de Antígenos de Linfócitos T/imunologia , Spiruroidea/imunologia , Transgenes/imunologiaRESUMO
Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.
Assuntos
Agrobacterium tumefaciens/fisiologia , Interações Hospedeiro-Patógeno , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Mapeamento de Interação de Proteínas/métodos , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting/métodos , Cromatografia de Afinidade/métodos , Vetores Genéticos/análise , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação/métodos , Microscopia Confocal/métodos , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas em Tandem/métodos , Nicotiana/metabolismoRESUMO
Adeno-associated virus 9 (AAV9) has been identified as one of the optimal gene transduction carriers for gene therapy. The aim of the present study was to determine the gene transfection efficiency and safety of an AAV9 vector produced using a recombinant baculovirus (rBac)based system. AAV9cytomegalovirus (CMV)-green fluorescent protein was produced using an rBac system and the resulting vector particles were injected intravenously into mice. Animals were sacrificed at 14, 21, 28, 35, 60, 90 and 120 days following injection. GFP expression in aortic vasculature and aortic plaques in C57/6B and apolipoprotein E/ mice was analyzed by fluorescence imaging and western blotting. In vivo analyses of biological markers of liver and heart damage, and renal function, as well as in vitro terminal deoxynucleotidyl transferase dUTP nick end labeling analysis were used to determine the toxicity of the AAV9 carrier. The findings of the present study demonstrated that AAV9 viral vectors packaged using the rBac system functioned appropriately in arteriosclerosis plaques. The CMV promoter significantly induced GFP expression in the vascular plaque in a time-dependent manner. AAV9CMV viral particles did not lead to heart, liver or renal damage and no change in apoptotic rate was identified. These findings indicated that AAV9-CMV may be effectively and safely used to transfect genes into atherosclerotic plaques.
Assuntos
Aterosclerose/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Placa Aterosclerótica/genética , Transfecção/métodos , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/patologia , Aterosclerose/terapia , Baculoviridae/genética , Citomegalovirus/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/patologia , Placa Aterosclerótica/terapia , Regiões Promotoras Genéticas , Transdução GenéticaRESUMO
Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.
Assuntos
Encéfalo/diagnóstico por imagem , Proteínas do Capsídeo/análise , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/análise , Radioisótopos do Iodo/administração & dosagem , Cintilografia/métodos , Aminopeptidases/metabolismo , Proteínas do Capsídeo/efeitos da radiação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Terapia Genética/métodos , Humanos , Masculino , Tomografia por Emissão de Pósitrons , Serina Proteases/metabolismo , Tripeptidil-Peptidase 1 , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40-50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.
Assuntos
Membrana Celular/química , DNA/química , Vetores Genéticos/análise , Substâncias Intercalantes/análise , Polímeros/análise , Cátions/análise , Cátions/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos/farmacologia , Células HEK293 , Humanos , Substâncias Intercalantes/farmacologia , Tamanho da Partícula , Polímeros/farmacologia , Porosidade , Propriedades de SuperfícieRESUMO
Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3'- and 5'- sequences adjacent to the integrated lentiviral vector.
Assuntos
DNA/análise , Reação em Cadeia da Polimerase/métodos , DNA/genética , Vetores Genéticos/análise , Vetores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lentivirus/genética , Retroviridae/genéticaRESUMO
Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.
Assuntos
Dependovirus/genética , Vetores Genéticos/análise , Genoma Viral , Reação em Cadeia da Polimerase , Primers do DNA/metabolismo , DNA de Cadeia Simples/análise , Eletroforese em Gel de Ágar , Células HEK293 , HumanosRESUMO
BACKGROUND: Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions. METHODS: Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 10(8) cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA. RESULTS: Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 10(12) VRX496 proviral copies were administered to these 65 subjects. CONCLUSIONS: Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors.
Assuntos
Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , HIV-1/genética , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Ensaio de Imunoadsorção Enzimática , Seguimentos , Vetores Genéticos/análise , HIV-1/fisiologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transdução Genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Carga Viral , Replicação ViralRESUMO
Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.
Assuntos
Adenoviridae/genética , DNA Viral/análise , Vetores Genéticos/análise , Genoma Viral , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Membrana Celular/virologia , DNA Viral/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Transdução GenéticaRESUMO
BACKGROUND: Lentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells. So far the most widely used method for concentration of lentiviral particles is ultracentrifugation (UC).An important feature of vectors derived from lentiviruses and prototypic gamma-retroviruses is that the host range can be altered by pseudotypisation. The most commonly used envelope protein for pseudotyping is the glycoprotein of the Vesicular Stomatitis Virus (VSV.G), which is also essential for successful concentration using UC. RESULTS: Here, we describe a purification method that is based on membrane adsorbers (MAs). Viral particles are efficiently retained by the anionic exchange MAs and can be eluted with a high-salt buffer. Buffer exchange and concentration is then performed by utilizing ultrafiltration (UF) units of distinct molecular weight cut off (MWCO). With this combined approach similar biological titers as UC can be achieved (2 to 5×109 infectious particles (IP)/ml). Lentiviral particles from small starting volumes (e.g. 40 ml) as well as large volumes (up to 1,000 ml) cell culture supernatant (SN) can be purified. Apart from LVs, vectors derived from oncoretroviruses can be efficiently concentrated as well. Importantly, the use of the system is not confined to VSV.G pseudotyped lenti- and retroviral particles and other pseudotypes can also be purified. CONCLUSIONS: Taken together the method presented here offers an efficient alternative for the concentration of lenti- as well as retroviral vectors with different pseudotypes that needs no expensive equipment, is easy to handle and can be used to purify large quantities of viral vectors within a short time.
Assuntos
Vetores Genéticos/isolamento & purificação , Lentivirus/isolamento & purificação , Retroviridae/isolamento & purificação , Ultrafiltração/métodos , Western Blotting , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/análise , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Ultracentrifugação/métodos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/isolamento & purificaçãoRESUMO
Safety and high transfection efficiency are the prerequisites for an ideal gene vector. Polyethylenimine (PEI), especially PEI 25k (25 kDa), is a well-known cationic gene carrier with high transfection efficiency. However, the high toxicity, depended on its molecular weight, has limited its use as a potential gene carrier. In our research, for the purpose of reducing the toxicity and increasing the transfection efficiency and further to inspect where the degradation of these biodegradable polymers take place would be more beneficial, in cytoplasm or in endocytic vesicles, two kinds of degradable polymers were synthesized. One is an acid-liable PEI derivate (PEI-GA) which was cross-linked by PEI 2k with glutadialdehyde (GA) through imine linkages and the other one (PEI-TEG) was cross-linked PEI 2k with modified triethyleneglycol (TEG) through biscarbamate linkages and can be degraded at neutral environment. By the use of a series of assay methods both in vitro and in vivo, the results showed that PEI-TEG was found to be biodegradable at neutral environment and exhibit high transfection ability with low toxicity, which indicated its potential as a candidate carrier for gene therapy.
Assuntos
Portadores de Fármacos/química , Vetores Genéticos/química , Implantes Absorvíveis , Aldeídos/química , Animais , Reagentes de Ligações Cruzadas/química , Desoxirribonuclease I/análise , Portadores de Fármacos/análise , Excipientes/química , Feminino , Vetores Genéticos/análise , Células HEK293 , Células Hep G2 , Humanos , Iminas/química , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Plasmídeos/química , Polietilenoglicóis/química , Polietilenos/química , Polímeros/análise , Polímeros/química , Polímeros/metabolismo , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , TransfecçãoRESUMO
Surface modification using poly(ethylene glycol) (PEG) is a widely used strategy to improve the biocompatibility of cationic polymer-based nonviral gene vectors (polyplexes). A novel method based on intravital real-time confocal laser scanning microscopy (IVRTCLSM) was applied to quantify the dynamic states of polyplexes in the bloodstream, thereby demonstrating the efficacy of PEGylation to prevent their agglomeration. Blood flow in the earlobe blood vessels of experimental animals was monitored in a noninvasive manner to directly observe polyplexes in the circulation. Polyplexes formed distinct aggregates immediately after intravenous injection, followed by interaction with platelets. To quantify aggregate formation and platelet interaction, the coefficient of variation and Pearson's correlation coefficient were adopted. In contrast, polyplex micelles prepared through self-assembly of plasmid DNA with PEG-based block catiomers had dense PEG palisades, revealing no formation of aggregates without visible interaction with platelets during circulation. This is the first report of in situ monitoring and quantification of the availability of PEGylation to prevent polyplexes from agglomeration over time in the blood circulation. This shows the high utility of IVRTCLSM in drug and gene delivery research.
Assuntos
Sistemas Computacionais , Pavilhão Auricular/irrigação sanguínea , Pavilhão Auricular/química , Vetores Genéticos/análise , Micelas , Polietilenoglicóis/análise , Animais , Circulação Sanguínea/fisiologia , Plaquetas/química , Pavilhão Auricular/efeitos dos fármacos , Feminino , Vetores Genéticos/administração & dosagem , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal/métodos , Polietilenoglicóis/administração & dosagemRESUMO
Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important.
Assuntos
Linhagem da Célula , Células Clonais/química , DNA Recombinante/análise , Marcadores Genéticos , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/química , Oligodesoxirribonucleotídeos/análise , Retroviridae/genética , Análise de Sequência de DNA/métodos , Animais , Distribuição Binomial , Separação Celular/métodos , Citometria de Fluxo/métodos , Terapia Genética/métodos , Vetores Genéticos/análise , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Transgenes , Integração ViralRESUMO
The pre-existing humoral and cellular immunity found in the great majority of the population raises concerns about the clinical efficacy and safety of vectors derived from ubiquitous human adenovirus serotypes. To alleviate these concerns, canine adenovirus type 2 vectors (CAV-2) were developed. Owing to their extraordinary neuronal tropism and efficient axonal retrograde transport, CAV-2 vectors hold great promise for the treatment of neurodegenerative diseases. The development and validation of a SYBR Green qPCR assay for determination of CAV-2 titers is reported in the present study. This method uses specific primers designed to amplify a small genomic fragment of CAV-2 structural genes (pVI-hexon). The method was accurate and reproducible as determined by the low intra- and inter-assay variability (<15% R.S.E.). It is sensitive and useful over a 5-log range (1 x 10(3) to 1 x 10(7)genome copies/reaction). The assay can be used to quantify purified vector samples as well as crude viral lysates. The titers obtained by qPCR correlated well with both, those obtained by OD(260) and TCID(50) as indicated by the high coefficients of determination obtained by regression analysis (r(2)>0.83). The development of this simple and rapid CAV-2 quantitation method should be helpful for process development and monitoring.
Assuntos
Adenovirus Caninos/isolamento & purificação , DNA Viral/análise , Vetores Genéticos/análise , Reação em Cadeia da Polimerase/métodos , Carga Viral/métodos , Adenovirus Caninos/genética , Animais , Linhagem Celular , Cães , Humanos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. MATERIALS AND METHODS: From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. RESULTS: In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. CONCLUSION: Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Animais , Linhagem da Célula , Células Cultivadas/transplante , Genes Reporter , Vetores Genéticos/análise , Sobrevivência de Enxerto , Macaca fascicularis , Osteoblastos/citologia , Células Estromais/transplante , Condicionamento Pré-Transplante , Transplante AutólogoRESUMO
Retroviral vectors have been widely used for research and clinical trials in gene therapy because of their high transduction efficiency. Retroviruses interact with target cells through their surface molecules (i.e., envelope proteins) and cellular receptors, which limit the susceptibility of target cells to retroviral vectors. Murine leukemia retrovirus (MuLV) pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G) overcomes the species barrier and is more resistant to mechanical and biochemical inactivation. A cell line producing VSV-G pseudotyped MuLV vector can be established by transfecting 293T cells expressing Gag, Pol, and VSV-G (293 GPG cell line) with a retroviral vector plasmid. Transduction potency of the resulting VSV-G pseudotyped MuLV retroviral supernatant can be quantified by titration, electron microscopy (EM), and the reverse transcriptase (RT) assay. These protocols provide methods to prepare and quantify a pseudotyped retroviral vector with high transduction rates for most types of target cells.