Accurate Quantification of AAV Vector Genomes by Quantitative PCR.

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.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

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)

Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses.

Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi- cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi- cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Small-scale protein expression in mammalian HEK293T cells Basic Protocol 2: Generation of baculovirus from Sf9 (insect) cells Alternate Protocol: Enumeration-free method for generating P2 viral stock Support Protocol 1: Small-scale transduction of HEK293T cells with P2 baculovirus Basic Protocol 3: Large-scale viral transduction of HEK293S GnTi- cells Support Protocol 2: Large-scale membrane preparation from HEK293S GnTi- cells Basic Protocol 4: Large-scale purification of membrane proteins from HEK293S GnTi- cells.

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes.

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 (∼30 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.

Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation.

BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.

Serum- and glucocorticoid-inducible kinase 1 activity reduces dendritic spines in dorsal hippocampus.

The hippocampus has a well-known role in mediating learning and memory, and its function can be directly regulated by both stress and glucocorticoid receptor activation. Hippocampal contributions to learning are thought to be dependent on changes in the plasticity of synapses within specific subregions, and these functional changes are accompanied by morphological changes in the number and shape of dendritic spines, the physical correlates of these glutamatergic synapses. Serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates dendritic spine morphology in the prefrontal cortex, and modulation of SGK1 expression in mouse hippocampus regulates learning. However, the role of SGK1 in dendritic spine morphology within the CA1 and dentate gyrus regions of the hippocampus are unknown. Thus, herpes simplex viral vectors expressing GFP and various SGK1 constructs, including wild type SGK1, a catalytically inactive version of SGK1 (K127Q), and a phospho-defective version of SGK1 (S78A), were infused into the hippocampus of adult mice and confocal fluorescent microscopy was used to visualize dendritic spines. We show that increasing expression of SGK1 in the dentate gyrus increased the total number of spines, driven primarily by an increase in mushroom spines, while decreasing SGK1 activity (K127Q) in the CA1 region increased the total number of dendritic spines, driven by a significant increase in mushroom and stubby spines. The differential effects of SGK1 in these regions may be mediated by the interactions of SGK1 with multiple pathways required for spine formation and stability. As the formation of mature synapses is a crucial component of learning and memory, this indicates that SGK1 is a potential target in the pathway underlying stress-associated changes in cognition and memory.

Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors.

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.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector.

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.

Genetic manipulation of specific neural circuits by use of a viral vector system.

To understand the mechanisms underlying higher brain functions, we need to analyze the roles of specific neuronal pathways or cell types forming the complex neural networks. In the neuroscience field, the transgenic approach has provided a useful gene engineering tool for experimental studies of neural functions. The conventional transgenic technique requires the appropriate promoter regions that drive a neuronal type-specific gene expression, but the promoter sequences specifically functioning in each neuronal type are limited. Previously, we developed novel types of lentiviral vectors showing high efficiency of retrograde gene transfer in the central nervous system, termed highly efficient retrograde gene transfer (HiRet) vector and neuron-specific retrograde gene transfer (NeuRet) vector. The HiRet and NeuRet vectors enable genetical manipulation of specific neural pathways in diverse model animals in combination with conditional cell targeting, synaptic transmission silencing, and gene expression systems. These newly developed vectors provide powerful experimental strategies to investigate, more precisely, the machineries exerting various neural functions. In this review, we give an outline of the HiRet and NeuRet vectors and describe recent representative applications of these viral vectors for studies on neural circuits.

Assessment of UV spectrophotometry for determination of plasmid DNA concentration in vector preparations for human gene therapy products.

The European Pharmacopoeia (Ph. Eur.) general chapter 5.14. Gene transfer medicinal products for human use suggests the use of absorbance measurements at 260 nm to determine the DNA concentration of plasmid vectors used for the preparation of gene therapy products for human use. An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to confirm the suitability of UV spectrophotometry for the quantification of plasmid vectors used in gene therapy (GT). Three Official Medicine Control Laboratories (OMCLs of the European OMCL Network) and members of the OMCL Working Group for GT products took part in the study, in which various types of spectrophotometers were assessed using common test samples. Results of the study demonstrated that UV spectrophotometry can be considered suitable for the quantification of plasmid DNA in GT products regardless of the instrument used.

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

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.

Recombinant adeno-associated virus serotype 9 in a mouse model of atherosclerosis: Determination of the optimal expression time in vivo.

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.

Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors.

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.

A bio-chemical application of N-GQDs and g-C3N4 QDs sensitized TiO2 nanopillars for the quantitative detection of pcDNA3-HBV.

Herein, TiO2 nanopillars (NPs)/N-doped graphene quantum dots (N-GQDs)/g-C3N4 QDs heterojunction efficiently suppressed the photogenerated charges recombination and improved photo-to-current conversion efficiency. The introduced N-GQDs and g-C3N4 QDs could result in more effective separation of the photogenerated charges, and thus produce a further increase of the photocurrent. TiO2 NPs/N-GQDs/g-C3N4 QDs were firstly applied as the photoactive materials for the fabrication of the biosensors, and the primers of pcDNA3-HBV were then adsorbed on the TiO2 NPs/N-GQDs/g-C3N4 QDs modified electrode under the activation of EDC/NHS. With increase of the pcDNA3-HBV concentration, the photocurrent reduced once the double helix between the primers and pcDNA3-HBV formed. The developed photoelectrochemical (PEC) biosensor showed a sensitive response to pcDNA3-HBV in a linear range of 0.01 fmol/L to 20nmol/L with a detection limit of 0.005 fmol/L under the optimal conditions. The biosensor exhibited high sensitivity, good selectivity, good stability and reproducibility.

Nanopartículas sólidas lipídicas para terapia génica / Solid lipid nanoparticles for gene therapy

La terapia génica consiste en la administración de ácidos nucleicos con el fin de modular la expresión de proteínas específicas y revertir así una enfermedad. Es una nueva área de la medicina con gran potencial para el tratamiento de enfermedades tanto hereditarias como adquiridas. Hasta ahora, los sistemas virales de administración de ácidos nucleicos han resultado eficaces, pero presentan importantes problemas de seguridad. Los vectores no virales, en cambio, son más seguros pero menos eficaces, aunque su eficacia ha aumentado significativamente en los últimos años. Esta revisión recoge la contribución de nuestro grupo de investigación al diseño de vectores no virales basados en nanopartículas sólidas lipídicas (SLNs) para terapia génica. Hemos estudiado la relación entre factores de la formulación con los procesos de internalización y disposición intracelular del material genético, que condicionan la eficacia de transfección, y por primera vez demostramos la capacidad de las SLNs para inducir la síntesis de una proteína tras su administración endovenosa a ratones. Esta revisión también recoge nuestros trabajos sobre la aplicación de las SLNs en el tratamiento de enfermedades infecciosas y enfermedades raras, como la retinosquisis juvenil ligada al cromosoma X, enfermedad en la que la retina está desestructurada debido a la deficiencia de la proteína retinosquisina. La administración de SLNs con el gen que codifica esta proteína en un modelo animal de esta enfermedad indujo la recuperación estructural de la retina. Los trabajos aquí recogidos muestran el gran potencial de las SLNs como sistemas de administración de ácidos nucleicos (AU)

Gene therapy is a rapidly advancing field with great potential for the treatment of genetic and acquired systemic diseases. This therapy requires the introduction of foreign genetic material in the target cells to modify a genetic sequence. Viral vectors are the most effective, but their application is limited by their immunogenicity, oncogenicity and the small size of the DNA they can transport. Non-viral vectors, however, are safer, lowered cost, and more reproducible and do not present DNA size limit. The main problem of nonviral systems is their low transfection efficiency, although it has improved during the last years. This review presents the contribution of our research group to the design and evaluation of SLNs based non-viral vectors for gene transfer. We report our studies about the relationship between formulation factors and cell uptake and intracellular trafficking of the genetic material, very important for transfection. We have shown, for the first time, the ability to induce transgene protein expression of the SLNs after endovenous administration to mice. This revision also reports our work about the potential application of SLNs to the treatment of infectious and rare diseases, as the X-linked juvenile retinoschisis, a retinal disorder due to a deficiency in the protein retinoschisin, and characterized by poor eyesight and degeneration of the retina. The intraocular injection of SLNs bearing the gene that encodes retinoschisin to diseased mice led to the partial recovery of the retina. All together, our results show the potential of SLNs as a system for gene delivery (AU)

Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane.

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.

Linear amplification mediated PCR--localization of genetic elements and characterization of unknown flanking DNA.

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.

Analysis of partial recombinants in lentiviral vector preparations.

The presence of replication-competent lentivirus (RCL) in lentiviral vector preparations is a major safety concern for clinical applications of such vectors. RCL are believed to emerge from rare recombinant vector genomes that are referred to as partial recombinants or Psi-Gag recombinants. To quantitatively determine the fraction of partial recombinants in lentiviral vector preparations and to analyze them at the DNA sequence level, we established a drug selection assay involving a lentiviral packaging construct containing a drug-resistance gene encoding blasticidin (BSD) resistance. Upon transduction of target cells, the BSD resistance gene confers BSD resistance to the transduced cells. The results obtained indicate that there were up to 156 BSD-resistant colonies in a total of 10(6) transducing vector particles. The predicted recombination events were verified by polymerase chain reaction using genomic DNA obtained from BSD-resistant cell clones and by DNA sequence analysis. In an attempt to reduce the emergence of partial recombinants, sequence overlaps between the packaging and the vector constructs were reduced by substituting the Rev response element (RRE) present in the vector construct using a heterologous RRE element derived from simian immunodeficiency virus (SIVmac239). The results obtained showed that a reduction of sequence overlaps resulted in an up to sevenfold reduction of the frequency of BSD-resistant colonies, indicating that the capacity to form partial recombinants was diminished.

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

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.

PCR-based detection of gene transfer vectors: application to gene doping surveillance.

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.